Agents modulating beta-catenin functions and methods thereof

ABSTRACT

Among other things, the present disclosure provides technologies for modulating functions of beta-catenin. In some embodiments, the present disclosure provides stapled peptides that interact with beta-catenin. In some embodiments, provided stapled peptides interact with beta-catenin at an Axin-binding site of beta-catenin. In some embodiments, the present disclosure provides compounds, compositions and methods for preventing and/or treating conditions, disorders and diseases that are associated with beta-catenin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Entry of International ApplicationNo. PCT/US18/50102, filed Sep. 7, 2018, which claims priority to U.S.Provisional Application No. 62/555,519, filed Sep. 7, 2017, the entiretyof each of which is incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 16, 2020, isnamed 2012675-0124_SL.txt and is 170,360 bytes in size.

BACKGROUND

Beta-catenin is a multifunctional protein and is involved in manybiological pathways and processes.

SUMMARY

Beta-catenin has many functions and regulates and coordinates manyprocesses, e.g., gene transcription, cell-cell adhesion, embryogenesis,cell growth, regeneration, etc. Among other things, beta-catenin playsimportant roles in the Wnt/beta-catenin pathway. Many conditions,disorders, and diseases, including a number of cancers (e.g.,hepatocellular carcinoma, colorectal carcinoma, lung cancer, malignantbreast tumors, ovarian and endometrial cancer, etc.), various forms ofheart diseases, etc., are associated with beta-catenin (e.g., itsabnormal levels, activities, localization, etc.).

Among other things, the present disclosure provides technologies (e.g.,compounds, compositions, methods, etc.) for modulating beta-cateninfunction. In some embodiments, such technologies are useful for, e.g.,preventing or treating beta-catenin associated conditions, disorders, ordiseases.

In some embodiments, the present disclosure encompasses the recognitionthat it can be beneficial to selectively or specifically modulate one ormore certain functions of beta-catenin, for example, functions thatinvolve an Axin binding site of beta-catenin. In some embodiments, suchfunctions involve interactions of Axin with beta-catenin. In someembodiments, the present disclosure provides technologies forselectively or specifically modulating beta-catenin functions. In someembodiments, provided technologies selectively or specifically modulatebeta-catenin functions involving one or more beta-catenin sites thatinteract with Axin. In some embodiments, provided technologiesselectively or specifically modulate beta catenin functions that involveinteractions between beta-catenin and Axin.

Among other things, the present disclosure provides agents, e.g.,stapled peptides, that physically interact with beta-catenin. In someembodiments, provided agents binds to beta-catenin at a site that Axinbinds to beta-catenin (e.g., at a site that overlaps with or isidentical to that at which Axin binds; alternatively or additionally, insome embodiments at a site with sufficient proximity to such Axinbinding site that the provided agent competes with Axin for binding tobeta-catenin). In some embodiments, provided agents interacts with someor all amino acid residues of beta-catenin that interact with Axin whenAxin binds to beta-catenin. In some embodiments, provided agents competewith Axin for binding to beta-catenin.

In some embodiments, provided agents are stapled peptides. In someembodiments, provided stapled peptides comprise a number of natural ornon-natural amino acid residues (e.g., 7-50, 10-25, 10-20, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, etc.), and one or more staples, each of which is independently alinker that can link one amino acid residue to another amino acidresidue and, as is understood by those skilled in the art, is not partof the peptide backbone.

In some embodiments, the present disclosure provides the insights thatstructural elements of staples (e.g., chemistry [e.g., hydrocarbon,non-hydrocarbon (e.g., comprising one or more heteroatoms orheteroatom-containing moieties such as amino, carbamate, etc.)],stereochemistry [e.g., stereochemistry of backbone atoms that staplesare connected to (e.g., if staples are connected to alpha-carbon atomsof amino acid residues, such carbon atoms being chiral (R S) orachiral)], positioning (to what amino acid residues/backbone atomsstaples are connected), sizes (length of staples), etc.), peptidesequences, lengths, and/or other modifications (e.g., incorporation ofunnatural amino acids, labels, targeting moieties [carbohydrate, proteinligand, etc.], etc.) can significantly impact properties and/oractivities, and can be employed to design stapled peptides havingsignificantly improved properties and/or activities (e.g., increasedsolubility, increased cell permeability, increased stability, increasedselectivity, lowered toxicity, increased activity, etc.).

Among other things, the present disclosure provides staples with variousstructural elements, and peptides that contain them. In someembodiments, a staple is a hydrocarbon staple. In some embodiments, astaple is a non-hydrocarbon staple in that it comprises one or moreheteroatoms. In some embodiments, a staple comprises an amino moiety(e.g., —N(R′)—, wherein R′ is as described in the present disclosure).In some embodiments, a staple comprises a carbamate moiety (e.g.,—N(R)—C(O)—O—, wherein R is as described in the present disclosure). Insome embodiments, a staple is a Pro-staple in that an end of the stapleis connected to a proline residue. In some embodiments, a staple is-L^(s)- as described in the present disclosure.

In some embodiments, provided stapled peptides comprising a staplecomprising an amino moiety or a carbamate moiety have improvedsolubility compared to an appropriate reference peptide (e.g., in someembodiments, peptides which are otherwise identical but do not containany staples or contain hydrocarbon staples instead of staples comprisingan amino or carbamate moiety). In some embodiments, provided peptidescomprising a staple comprising an amino moiety or a carbamate moietyhave increased cell permeability compared to an appropriate referencepeptide. In some embodiments, provided peptides comprising a staplecomprising an amino moiety or a carbamate moiety have increasedactivities, e.g., increased inhibition of gene expression, cell growth,etc.

In some embodiments, a staple connects amino acid residue i and i+m(wherein each of i and m is independently as described in the presentdisclosure), and the connecting atoms at amino acid residue i (C) and atamino acid residue i+m (C^(i+m)) are independently chiral and achiral,and if chiral, are independently racemic, R or S. In some embodiments,both C^(i) and C^(i+m) are carbon atoms. In some embodiments, C^(i) isachiral and C^(i+m) chiral. In some embodiments, C^(i) is a chiral andC^(i+m) is R. In some embodiments, C^(i) is a chiral and C^(i+m) is S.In some embodiments, C^(i) is chiral and C^(i+m) achiral. In someembodiments, C^(i) is R and C^(i+m) achiral. In some embodiments, C^(i)is S and C^(i+m) achiral. In some embodiments, C^(i) is R and C^(i+m) isR. In some embodiments, C^(i) is R and C^(i+m) is S. In someembodiments, C^(i) is S and C^(i+m) is R. In some embodiments, C^(i) isS and C^(i+m) is S. In some embodiments, controlling chemistry and/orstereochemistry significantly improves yields and/or purity of preparedstapled peptides, and/or properties and activities of provided stapledpeptides.

In some embodiments, the present disclosure provides a peptidecomprising:[X¹]_(p1)[X²]_(p2)—X³X⁴X⁵X⁶X⁷X⁸X⁹X¹⁰—[X¹¹]_(p11)[X¹²]_(p12)[X¹³]_(p13),wherein:

each of p1, p2, p11, p12 and p13 is independently 0 or 1;

each of X, X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², and X¹³ isindependently an amino acid residue;

at least two of X, X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹²,and X¹³ comprise side chains that are optionally linked together to forma staple.

In some embodiments, p1 is 0. In some embodiments, p1 is 1. In someembodiments, p2 is 0. In some embodiments, p2 is 1.

In some embodiments, p11 is 0. In some embodiments, p11 is 1. In someembodiments, p12 is 0. In some embodiments, p12 is 1. In someembodiments, p13 is 0. In some embodiments, p13 is 1.

In some embodiments, the present disclosure provides a peptidecomprising a staple L^(s), wherein L^(s) is an optionally substituted,bivalent C₁₋₅₀ aliphatic group wherein one or more methylene units ofthe aliphatic group are optionally and independently replaced with—C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—,—S(O)₂N(R′)—, —C(O)S—, or —C(O)O—; each -Cy- is independently anoptionally substituted bivalent group selected from a C₃₋₂₀cycloaliphatic ring, a C₆₋₂₀ aryl ring, a 5-20 membered heteroaryl ringhaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ringhaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon;

each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R;

each R is independently —H, or an optionally substituted group selectedfrom C1-30 aliphatic, C₁₋₃₀ heteroaliphatic having 1-10 heteroatomsindependently selected from oxygen, nitrogen, sulfur, phosphorus andsilicon, C₆₋₃₀ aryl, C₆₋₃₀ arylaliphatic, C₆₋₃₀ arylheteroaliphatichaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon, or

two R groups are optionally and independently taken together to form acovalent bond, or:

two or more R groups on the same atom are optionally and independentlytaken together with the atom to form an optionally substituted, 3-30membered, monocyclic, bicyclic or polycyclic ring having, in addition tothe atom, 0-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon; or

two or more R groups on two or more atoms are optionally andindependently taken together with their intervening atoms to form anoptionally substituted, 3-30 membered, monocyclic, bicyclic orpolycyclic ring having, in addition to the intervening atoms, 0-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon.

In some embodiments, the present disclosure provides a peptide havingthe structure:

or a salt thereof, wherein

each of R^(a), R¹, R², R³, and R⁴ is independently R′;

R^(b) is R′, —OR′ or —N(R′)₂;

each of X is independently an amino acid residue;

each of a, b, c, s, and d is independently 1-20;

each of C¹ and C² is independently a carbon atom;

each L^(s) is independently -L^(s1)-L^(s2)-L^(s3)-, wherein L^(s1) isbonded to C¹ and L^(s3) is bonded to C²;

each of L^(s1), L^(s2), and L^(s3) is independently L;

each L is independently a covalent bond, or an optionally substituted,bivalent C₁-C₂₀ aliphatic group wherein one or more methylene units ofthe aliphatic group are optionally and independently replaced with—C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—,—S(O)₂N(R′)—, —C(O)S—, or —C(O)O—;

each -Cy- is independently an optionally substituted bivalent groupselected from a C₃₋₂₀ cycloaliphatic ring, a C₆₋₂₀ aryl ring, a 5-20membered heteroaryl ring having 1-10 heteroatoms independently selectedfrom oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20membered heterocyclyl ring having 1-10 heteroatoms independentlyselected from oxygen, nitrogen, sulfur, phosphorus and silicon;

each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R;

each R is independently —H, or an optionally substituted group selectedfrom C₁₋₃₀ aliphatic, C₁₋₃₀ heteroaliphatic having 1-10 heteroatomsindependently selected from oxygen, nitrogen, sulfur, phosphorus andsilicon, C₆₋₃₀ aryl, C₆₋₃₀ arylaliphatic, C₆₋₃₀ arylheteroaliphatichaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon, or

two R groups are optionally and independently taken together to form acovalent bond, or:

two or more R groups on the same atom are optionally and independentlytaken together with the atom to form an optionally substituted, 3-30membered, monocyclic, bicyclic or polycyclic ring having, in addition tothe atom, 0-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon; or

two or more R groups on two or more atoms are optionally andindependently taken together with their intervening atoms to form anoptionally substituted, 3-30 membered, monocyclic, bicyclic orpolycyclic ring having, in addition to the intervening atoms, 0-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon.

In some embodiments, R^(a) is R′, wherein R′ is as described in thepresent disclosure. In some embodiments, R^(a) is —H. In someembodiments, R^(a) is R—C(O)—.

In some embodiments, X is a residue of an amino acid of formula A-I. Insome embodiments, X is a residue of an amino acid of formula A-II. Insome embodiments, X is a residue of an amino acid of formula A-III.

In some embodiments, a is 1. In some embodiments, a is 2. In someembodiments, a is 3. In some embodiments, a is 4. In some embodiments, ais 5. In some embodiments, a is 6. In some embodiments, a is 7. In someembodiments, a is 8. In some embodiments, a is 9. In some embodiments, ais 10. In some embodiments, a is 11. In some embodiments, a is 12. Insome embodiments, a is 13. In some embodiments, a is 14. In someembodiments, a is 15. In some embodiments, a is 16. In some embodiments,a is 17. In some embodiments, a is 18. In some embodiments, a is 19. Insome embodiments, a is 20.

In some embodiments, R¹ is R′ as described in the present disclosure. Insome embodiments, R¹ is R as described in the present disclosure. Insome embodiments, R¹ is —H. In some embodiments, R¹ is not H. In someembodiments, R¹ and R′ of a —N(R′)— or —N(R′)—C(O)O— moiety of L^(s) orL^(s1) are R and are taken together with their intervening atoms to forman optionally substituted ring as described in the present disclosure.

In some embodiments, R² is R′ as described in the present disclosure. Insome embodiments, R² is R as described in the present disclosure. Insome embodiments, R² is —H. In some embodiments, R² is not H. In someembodiments, R¹ and R′ of a —N(R′)— or —N(R′)—C(O)O— moiety of L^(s) orL^(s3) are R and are taken together with their intervening atoms to forman optionally substituted ring as described in the present disclosure.

In some embodiments, R³ is R′ as described in the present disclosure. Insome embodiments, R³ is R as described in the present disclosure. Insome embodiments, R³ is —H. In some embodiments, R³ is not H.

In some embodiments, R⁴ is R′ as described in the present disclosure. Insome embodiments, R⁴ is R as described in the present disclosure. Insome embodiments, R⁴ is —H. In some embodiments, R⁴ is not H.

In some embodiments, C¹ is achiral. In some embodiments, C¹ is chiral.In some embodiments, C¹ is R. In some embodiments, C¹ is S.

In some embodiments, C² is achiral. In some embodiments, C² is chiral.In some embodiments, C² is R. In some embodiments, C² is S.

In some embodiments, b is 2-11. In some embodiments, b is 2. In someembodiments, b is 3. In some embodiments, b is 4. In some embodiments, bis 5. In some embodiments, b is 6. In some embodiments, b is 7. In someembodiments, b is 8. In some embodiments, b is 9. In some embodiments, bis 10. In some embodiments, b is 11.

In some embodiments, c is 1. In some embodiments, c is 2. In someembodiments, c is 3. In some embodiments, c is 4. In some embodiments, cis 5. In some embodiments, c is 6. In some embodiments, c is 7. In someembodiments, c is 8. In some embodiments, c is 9. In some embodiments, cis 10. In some embodiments, c is 11. In some embodiments, c is 12. Insome embodiments, c is 13. In some embodiments, c is 14. In someembodiments, c is 15. In some embodiments, c is 16. In some embodiments,c is 17. In some embodiments, c is 18. In some embodiments, c is 19. Insome embodiments, c is 20.

In some embodiments, s is 1-5. In some embodiments, s is 1. In someembodiments, s is 2. In some embodiments, s is 3. In some embodiments, sis 4. In some embodiments, s is 5.

In some embodiments, d is 1. In some embodiments, d is 2. In someembodiments, d is 3. In some embodiments, d is 4. In some embodiments, dis 5. In some embodiments, d is 6. In some embodiments, d is 7. In someembodiments, d is 8. In some embodiments, d is 9. In some embodiments, dis 10. In some embodiments, d is 11. In some embodiments, d is 12. Insome embodiments, d is 13. In some embodiments, d is 14. In someembodiments, d is 15. In some embodiments, d is 16. In some embodiments,d is 17. In some embodiments, d is 18. In some embodiments, d is 19. Insome embodiments, d is 20.

In some embodiments, R^(b) is R′ as described in the present disclosure.In some embodiments, R^(b) is R as described in the present disclosure.In some embodiments, R^(b) is —H. In some embodiments, R^(b) is —OR′wherein R′ is as described in the present disclosure. In someembodiments, R^(b) is —OH. In some embodiments, R^(b) is —N(R′)₂,wherein each R′ is independently as described in the present disclosure.In some embodiments, R^(b) is —NH(R′) wherein R′ is independently asdescribed in the present disclosure.

In some embodiments, the present disclosure provides a stapled peptidecomprising a staple having the structure of L^(s). In some embodiments,the present disclosure provides a stapled peptide comprising a staplehaving the structure of L^(s), wherein:

L^(s) is -L^(s1)-L^(s2)-L^(s3)-;

one end of L^(s) is connected to an atom A^(n1) of the peptide backbone,wherein A^(n1) is bonded to R¹;

one end of L^(s) is connected to an atom A^(n2) of the peptide backbone,wherein A² is bonded to R²;

each of R¹ and R² is independently R′;

there are m amino acid residues between the amino acid residuecomprising A^(n1) and the amino acid residue comprising A^(n2), notincluding the amino acid residue comprising A^(n1) and the amino acidresidue comprising A^(n2);

m is an integer of 1-12; and

wherein each other variable is independently as described in the presentdisclosure.

In some embodiments, A^(n1) is a carbon atom. In some embodiments, R¹bonded to A^(n1) and R′ of a —N(R′)— or —N(R′)—C(O)O— moiety of L^(s)are R and are taken together with their intervening atoms to form anoptionally substituted ring as described in the present disclosure. Insome embodiments, A^(n1) is achiral. In some embodiments, A^(n1) ischiral. In some embodiments, A^(n1) is R. In some embodiments, A^(n1) isS.

In some embodiments, A² is a carbon atom. In some embodiments, R² bondedto A^(n1) and R′ of a —N(R′)— or —N(R′)—C(O)O— moiety of L^(s) are R andare taken together with their intervening atoms to form an optionallysubstituted ring as described in the present disclosure. In someembodiments, A^(n2) is achiral. In some embodiments, A^(n2) is chiral.In some embodiments, A² is R. In some embodiments, A² is S.

In some embodiments, m is 1. In some embodiments, m is 2. In someembodiments, m is 3. In some embodiments, m is 4. In some embodiments, mis 5. In some embodiments, m is 6. In some embodiments, m is 7. In someembodiments, m is 8. In some embodiments, m is 9. In some embodiments, mis 10. In some embodiments, m is 11. In some embodiments, m is 12.

In some embodiments, provided agents, e.g., stapled peptides, areoptionally conjugated with a second entity, e.g., a targeting moiety(e.g., a carbohydrate, a receptor ligand, etc.), a second peptide, etc.In some embodiments, provided peptides are conjugated to one or moreligands for targeted delivery to cells expressing receptors to which theligands bind to. In some embodiments, provided agents are conjugated toone or more second entities that have an enzymatic activity, or ligandsfor proteins that have an enzymatic activity (e.g., E3 ubiquitinligase).

In some embodiments, provided agents, e.g., stapled peptides, have lowertoxicity compared to an appropriate reference peptide (e.g., a peptidehaving the same sequence but lacking a staple or having a staple thatdiffers in one or more features (e.g., chemistry [e.g., presence orabsence, and/or number and/or type of heteroatoms, degree of saturation,etc.], stereochemistry, length, etc.). Among other things, asdemonstrated in the present disclosure, in some embodiments providedpeptides have low cytotoxicity, and in particular low non-specificcytotoxicity, as compared to an appropriate reference peptide (e.g., incertain particular embodiments, an otherwise identical stapled peptidehaving a hydrocarbon staple).

In some embodiments, provided agents, e.g., stapled peptides, haveunexpected selectivity and/or specificity for modulating beta-cateninfunctions and/or Wnt pathway compared to other one or more comparablereference agents.

In some embodiments, provided agents, e.g., stapled peptides,selectively interact with Axin-interacting sites of beta-catenin andmodulate beta-catenin interactions with other entities (e.g., proteins,small molecules, etc.) at such Axin-interacting sites. As demonstratedin the present disclosure, in some embodiments, provided agents, e.g.,stapled peptides, can selectively disrupt beta-catenin interactions atAxin sites without significantly impacting interactions atBCL9-interacting sites of beta-catenin. Technologies for assessingselectivity are widely known in the art and can be utilized inaccordance with the present disclosure, e.g., certain fluorescenceassays described in the present disclosure.

In some embodiments, the present disclosure provides pharmaceuticalcompositions comprising a provided agent, e.g., a stapled peptide, and apharmaceutically acceptable carrier.

In some embodiments, the present disclosure provides technologies formodulating one or more beta-catenin functions. In some embodiments, thepresent disclosure provides agents, e.g., stapled peptides, andcompositions thereof for modulating beta-catenin functions. In someembodiments, the present disclosure provides technologies for inhibitingaberrant beta-catenin activities. As appreciated by those skilled in theart, beta-catenin plays important roles in Wnt signaling pathways andother biological pathways. In some embodiments, the present disclosureprovides technologies for modulating Wnt signaling pathway. In someembodiments, the present disclosure provides technologies for inhibitingaberrant Wnt signaling. In some embodiments, the present disclosureprovides technologies for modulating expression of a nucleic acidsequence in a system, comprising contacting a system comprisingbeta-catenin a provided stapled peptide, wherein expression of thenucleic acid sequence is associated with beta-catenin. In someembodiments, the present disclosure provides technologies for modulatinglevel of a product encoded by a nucleic acid sequence in a system,comprising contacting a system comprising beta-catenin a providedpeptide, wherein level of a product encoded by a nucleic acid sequenceis associated with beta-catenin.

In some embodiments, the present disclosure provides methods forpreventing and/or treating a condition, disorder, or diseases associatedwith beta-catenin. In some embodiments, the present disclosure providesmethods for preventing and/or treating a condition, disorder, ordiseases associated with Wnt signaling. In some embodiments, providedmethods comprise administering to a subject susceptible to or sufferingfrom a condition, disorder or disease associated with beta-cateninand/or Wnt signaling. In some embodiments, a condition, disorder, ordisease is cancer.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Provided agents can bind to beta-catenin. FIG. 1 depictsexemplary beta-catenin binding data from a competition fluorescencepolarization assay. Peptide solutions were prepared in buffer (50 mMTris pH 8.0, 250 mM NaCl, 2% glycerol, 0.5 mM EDTA, 0.02% w/v sodiumazide) using a 3-fold serial dilution from 5 μM. Probe solution (15 nMfull-length 13-Catenin, 20 nM FITC labeled peptide(FITC-PEG1-PQ-S5-ILD-S5-HVRRVWR (SEQ ID NO: 1) (hydrocarbon stapleformed by two S5 via olefin metathesis)) in buffer) was prepared andincubated for 5 minutes, then 40 μL per well plated in a blackpolystyrene 384-well plate (Corning). Equal volume of the titratedpeptide was added to the plate and incubated protected from light for 15minutes prior to read. Reads were performed on a Spectramax M5(Molecular Devices) in duplicate.

FIG. 2. Provided agents are active in cells. FIG. 2 depicts exemplarydata from TCF/LEF reporter assays. Y-axes illustrate luminescence, andX-axes illustrate concentrations of peptides. Inhibition of TCF/LEFReporter Activity by FP0025c and FPO217c Isomer 2. TCF/LEF Luciferasereporter HEK293 cell lines (BPS Bioscience) were treated with dilutionseries of FP0025c and FP00217c Isomer 2 for 18 hours. 300 ng/mL of Wnt3a(Peprotech) was added to the cells for the final 6 hours of incubation.Luciferase activity was measured using Bright-Glo Luciferase Assay(Promega) according to manufacturer's protocol.

FIG. 3. Provided agents modulate gene expression in cells. FIG. 3illustrates modulation of gene expression by an exemplary stapledpeptide. DLD-1 cells were treated with a dilution series of FPO217cisomer 2 for 18 hours. Total RNA was extracted using the RNeasy Plus kit(Qiagen) according to manufacturer's protocols, and reverse transcribedto cDNA using SuperScript Vilo IV master mix (ThermoFisher Scientific).Gene expression levels were determined by qPCR using Taqman probes(Applied Biosciences) and Taqman Advanced Fast Master Mix (AppliedBiosciences) on a QuantStudio 3 (Applied Biosciences). Relativeexpression was quantified using delta Ct method. For each group, fromleft to right, Axin 2, LEF1, Cyclin D, LRP6 and c-myc.

FIG. 4. Provided agents modulate gene expression in cells. FIG. 4illustrates modulation of gene expression by an exemplary stapledpeptide. HCT-116 cells were treated with a dilution series of FPO217cisomer 2 for 18 hours. Total RNA was extracted using the RNeasy Plus kit(Qiagen) according to manufacturer's protocols, and reverse transcribedto cDNA using SuperScript Vilo IV master mix (ThermoFisher Scientific).Gene expression levels were determined by qPCR using Taqman probes(Applied Biosciences) and Taqman Advanced Fast Master Mix (AppliedBiosciences) on a QuantStudio 3 (Applied Biosciences). Relativeexpression was quantified using delta Ct method. For each group, fromleft to right, Axin 2, VEGF, Cyclin D, LRP6 and c-myc.

FIG. 5. Provided agents can selectively modulate gene expression. DLD-1cells were treated with either 3 uM or 10 uM of each compound for 18hours. Total RNA was extracted using the RNeasy Plus kit (Qiagen)according to manufacturer's protocols, and reverse transcribed to cDNAusing SuperScript Vilo IV master mix (ThermoFisher Scientific). Geneexpression levels were determined by qPCR using Taqman probes (AppliedBiosciences) and Taqman Advanced Fast Master Mix (Applied Biosciences)on a QuantStudio 3 (Applied Biosciences). Relative expression wasquantified using delta Ct method. For each group, from left to right:Axin2, LEF1 and Cyclin D.

FIG. 6. Exemplary pharmacokinetic properties. Peptides were formulatedin 10% DMSO:90% saline and dosed by IV at 0.5 mg/kg per compound inthree male Sprague-Dawley rats. Serial bleed time-points were taken at 2min, 6 min, 10 min, 15 min, 30 min, 1, 2, 4, 6, 8, 12 and 24 h andanalyzed by quantitative LC/MS using a Thermo Q-Exactive Focus LC/MS/MS.Samples were prepared by protein precipitation with MeOH. Data were fitto a two-compartment model.

FIG. 7. Provided agents selectively disrupts interactions with Axin. Insome embodiments, provided agents, e.g., stapled peptides, selectivelydisrupts interactions at one or more beta-catenin sites that interactwith Axin over interactions at one or more beta-catenin sites thatinteract with BCL9. As illustrated in Panel A, FPO217c isomer 2 andFP0597c displaced a labeled Axin site probe. They, as shown in Panel B,did not displace the labeled BCL9 site probe but FP0650c (stapledpeptides designed to interact with one or more beta-catenin sites thatinteract with BCL9) did. BCL9 Competition FP assay: Peptide solutionswere prepared in buffer (50 mM Tris pH 8.0, 250 mM NaCl, 2% glycerol,0.5 mM EDTA, 0.02% w/v sodium azide) using a 3-fold serial dilution from10 μM. Probe solution (250 nM full-length beta-catenin, 20 nM 5FAMlabeled peptide in buffer) was prepared and 40 μL per well plated in ablack polystyrene 384-well plate (Corning). Equal volume of the titratedpeptide was added to the plate and incubated protected from light for 15minutes prior to read. Reads were performed on a Spectramax M5(Molecular Devices) in duplicate. Probe:Ac-Leu-Ser-Gln-Glu-Gln-Leu-Glu-His-Arg-Glu-Arg-Ser-Leu-Gln-Thr-Leu-Arg-Asp-Ile-Gln-Arg-nLeu-Leu-2NapA-bala-bala-Lys5FAM-NH2(SEQ ID NO: 2) (from Biochemistry, 2009, 48 (40), pp 9534-9541). AxinCompetition FP assay: Peptide solutions were prepared in buffer (50 mMTris pH 8.0, 250 mM NaCl, 2% glycerol, 0.5 mM EDTA, 0.02% w/v sodiumazide) using a 3-fold serial dilution from 5 μM. Probe solution (15 nMfull-length beta-catenin, 20 nM FITC labeled peptide in buffer) wasprepared and incubated for 5 minutes, then 40 μL per well plated in ablack polystyrene 384-well plate (Corning). Equal volume of the titratedpeptide was added to the plate and incubated protected from light for 15minutes prior to read. Reads were performed on a Spectramax M5(Molecular Devices) in duplicate. Probe: FITC-StAx-33 from Grossmann etal. PNAS 109 17942-17947.

FIG. 8. Exemplary results of various olefin metathesis methods. (A)Grubbs I, one treatment in DCE, at 40° C., 2 hrs. (B) Grubbs II, onetreatment in DCE, at 40° C., 2 hrs. (C) Hoveyda-Grubbs I, one treatmentin DCE, at 40° C., 2 hrs. (D) Hoveyda-Grubbs II, one treatment in DCE,at 60° C., 2 hrs.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS 1. Definitions

As used herein, the following definitions shall apply unless otherwiseindicated. For purposes of this disclosure, the chemical elements areidentified in accordance with the Periodic Table of the Elements, CASversion, Handbook of Chemistry and Physics, 75^(th) Ed. Additionally,general principles of organic chemistry are described in “OrganicChemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999,and “March's Advanced Organic Chemistry”, 5^(th) Ed., Ed.: Smith, M. B.and March, J., John Wiley & Sons, New York: 2001.

Administration: As used herein, the term “administration” typicallyrefers to the administration of a composition to a subject or system.Those of ordinary skill in the art will be aware of a variety of routesthat may, in appropriate circumstances, be utilized for administrationto a subject, for example a human. For example, in some embodiments,administration may be ocular, oral, parenteral, topical, etc. In someparticular embodiments, administration may be bronchial (e.g., bybronchial instillation), buccal, dermal (which may be or comprise, forexample, one or more of topical to the dermis, intradermal, interdermal,transdermal, etc), enteral, intra-arterial, intradermal, intragastric,intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal,intravenous, intraventricular, within a specific organ (e. g.,intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual,topical, tracheal (e.g., by intratracheal instillation), vaginal,vitreal, etc. In some embodiments, administration may involve dosingthat is intermittent (e.g., a plurality of doses separated in time)and/or periodic (e.g., individual doses separated by a common period oftime) dosing. In some embodiments, administration may involve continuousdosing (e.g., perfusion) for at least a selected period of time.

Agent: In general, the term “agent”, as used herein, may be used torefer to a compound or entity of any chemical class including, forexample, a polypeptide, nucleic acid, saccharide, lipid, small molecule,metal, or combination or complex thereof. In appropriate circumstances,as will be clear from context to those skilled in the art, the term maybe utilized to refer to an entity that is or comprises a cell ororganism, or a fraction, extract, or component thereof. Alternatively oradditionally, as context will make clear, the term may be used to referto a natural product in that it is found in and/or is obtained fromnature. In some instances, again as will be clear from context, the termmay be used to refer to one or more entities that is man-made in that itis designed, engineered, and/or produced through action of the hand ofman and/or is not found in nature. In some embodiments, an agent may beutilized in isolated or pure form; in some embodiments, an agent may beutilized in crude form. In some embodiments, potential agents may beprovided as collections or libraries, for example that may be screenedto identify or characterize active agents within them. In some cases,the term “agent” may refer to a compound or entity that is or comprisesa polymer; in some cases, the term may refer to a compound or entitythat comprises one or more polymeric moieties. In some embodiments, theterm “agent” may refer to a compound or entity that is not a polymerand/or is substantially free of any polymer and/or of one or moreparticular polymeric moieties. In some embodiments, the term may referto a compound or entity that lacks or is substantially free of anypolymeric moiety. In some embodiments, an agent is a compound. In someembodiments, an agent is a stapled peptide.

Aliphatic: As used herein, “aliphatic” means a straight-chain (i.e.,unbranched) or branched, substituted or unsubstituted hydrocarbon chainthat is completely saturated or that contains one or more units ofunsaturation, or a substituted or unsubstituted monocyclic, bicyclic, orpolycyclic hydrocarbon ring that is completely saturated or thatcontains one or more units of unsaturation, or combinations thereof.Unless otherwise specified, aliphatic groups contain 1-100 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-20aliphatic carbon atoms. In other embodiments, aliphatic groups contain1-10 aliphatic carbon atoms. In other embodiments, aliphatic groupscontain 1-9 aliphatic carbon atoms. In other embodiments, aliphaticgroups contain 1-8 aliphatic carbon atoms. In other embodiments,aliphatic groups contain 1-7 aliphatic carbon atoms. In otherembodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. Instill other embodiments, aliphatic groups contain 1-5 aliphatic carbonatoms, and in yet other embodiments, aliphatic groups contain 1, 2, 3,or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but arenot limited to, linear or branched, substituted or unsubstituted alkyl,alkenyl, alkynyl groups and hybrids thereof.

Alkenyl: As used herein, the term “alkenyl” refers to an aliphaticgroup, as defined herein, having one or more double bonds.

Alkenylene: The term “alkenylene” refers to a bivalent alkenyl group.

Alkyl: As used herein, the term “alkyl” is given its ordinary meaning inthe art and may include saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkylsubstituted alkyl groups. In some embodiments, alkyl has 1-100 carbonatoms. In certain embodiments, a straight chain or branched chain alkylhas about 1-20 carbon atoms in its backbone (e.g., C₁-C₂₀ for straightchain, C₂-C₂₀ for branched chain), and alternatively, about 1-10. Insome embodiments, cycloalkyl rings have from about 3-10 carbon atoms intheir ring structure where such rings are monocyclic, bicyclic, orpolycyclic, and alternatively about 5, 6 or 7 carbons in the ringstructure. In some embodiments, an alkyl group may be a lower alkylgroup, wherein a lower alkyl group comprises 1-4 carbon atoms (e.g.,C₁-C₄ for straight chain lower alkyls).

Alkylene: The term “alkylene” refers to a bivalent alkyl group.

Amino acid: In its broadest sense, as used herein, refers to anycompound and/or substance that can be incorporated into a polypeptidechain, e.g., through formation of one or more peptide bonds. In someembodiments, an amino acid comprising an amino group and an a carboxylicacid group. In some embodiments, an amino acid has the structure ofNH(R^(a1))-L^(a1)-C(R^(a2))(R^(a3))-L^(a2)-COOH, wherein each variableis independently as described in the present disclosure. In someembodiments, an amino acid has the general structure NH(R′)—C(R′)₂—COOH,wherein each R′ is independently as described in the present disclosure.In some embodiments, an amino acid has the general structureH₂N—C(R′)₂—COOH, wherein R′ is as described in the present disclosure.In some embodiments, an amino acid has the general structureH₂N—C(H)(R′)—COOH, wherein R′ is as described in the present disclosure.In some embodiments, an amino acid is a naturally-occurring amino acid.In some embodiments, an amino acid is a non-natural amino acid; in someembodiments, an amino acid is a D-amino acid; in some embodiments, anamino acid is an L-amino acid. “Standard amino acid” refers to any ofthe twenty standard L-amino acids commonly found in naturally occurringpeptides. “Nonstandard amino acid” refers to any amino acid, other thanthe standard amino acids, regardless of whether it is preparedsynthetically or obtained from a natural source. In some embodiments, anamino acid, including a carboxy- and/or amino-terminal amino acid in apolypeptide, can contain a structural modification as compared with thegeneral structure above. For example, in some embodiments, an amino acidmay be modified by methylation, amidation, acetylation, pegylation,glycosylation, phosphorylation, and/or substitution (e.g., of the aminogroup, the carboxylic acid group, one or more protons, one or morehydrogens, and/or the hydroxyl group) as compared with the generalstructure. In some embodiments, such modification may, for example,alter the circulating half-life of a polypeptide containing the modifiedamino acid as compared with one containing an otherwise identicalunmodified amino acid. In some embodiments, such modification does notsignificantly alter a relevant activity of a polypeptide containing themodified amino acid, as compared with one containing an otherwiseidentical unmodified amino acid. As will be clear from context, in someembodiments, the term “amino acid” may be used to refer to a free aminoacid; in some embodiments it may be used to refer to an amino acidresidue of a polypeptide.

Analog: As used herein, the term “analog” refers to a substance thatshares one or more particular structural features, elements, components,or moieties with a reference substance. Typically, an “analog” showssignificant structural similarity with the reference substance, forexample sharing a core or consensus structure, but also differs incertain discrete ways. In some embodiments, an analog is a substancethat can be generated from the reference substance, e.g., by chemicalmanipulation of the reference substance. In some embodiments, an analogis a substance that can be generated through performance of a syntheticprocess substantially similar to (e.g., sharing a plurality of stepswith) one that generates the reference substance. In some embodiments,an analog is or can be generated through performance of a syntheticprocess different from that used to generate the reference substance.

Animal: As used herein refers to any member of the animal kingdom. Insome embodiments, “animal” refers to humans, of either sex and at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, insects, and/or worms. In someembodiments, an animal may be a transgenic animal, geneticallyengineered animal, and/or a clone.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%%, 15%, 14%, 13%, %12%, 11%, 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Aryl: The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” “aryloxyalkyl,” etc. refers to monocyclic,bicyclic or polycyclic ring systems having a total of five to thirtyring members, wherein at least one ring in the system is aromatic. Insome embodiments, an aryl group is a monocyclic, bicyclic or polycyclicring system having a total of five to fourteen ring members, wherein atleast one ring in the system is aromatic, and wherein each ring in thesystem contains 3 to 7 ring members. In some embodiments, an aryl groupis a biaryl group. The term “aryl” may be used interchangeably with theterm “aryl ring.” In certain embodiments of the present disclosure,“aryl” refers to an aromatic ring system which includes, but not limitedto, phenyl, biphenyl, naphthyl, binaphthyl, anthracyl and the like,which may bear one or more substituents. In some embodiments, alsoincluded within the scope of the term “aryl,” as it is used herein, is agroup in which an aromatic ring is fused to one or more non-aromaticrings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, ortetrahydronaphthyl, and the like, where a radical or point of attachmentis on an aryl ring.

Associated with: Two events or entities are “associated” with oneanother, as that term is used herein, if the presence, level and/or formof one is correlated with that of the other. For example, a particularentity (e.g., nucleic acid (e.g., genomic DNA, transcripts, mRNA, etc.),polypeptide, genetic signature, metabolite, microbe, etc.) is consideredto be associated with a particular disease, disorder, or condition, ifits presence, level and/or form correlates with incidence of and/orsusceptibility to the disease, disorder, or condition (e.g., across arelevant population).

Carrier: as used herein, refers to a diluent, adjuvant, excipient, orvehicle with which a composition is administered. In some exemplaryembodiments, carriers can include sterile liquids, such as, for example,water and oils, including oils of petroleum, animal, vegetable orsynthetic origin, such as, for example, peanut oil, soybean oil, mineraloil, sesame oil and the like. In some embodiments, carriers are orinclude one or more solid components.

Comparable: As used herein, the term “comparable” refers to two or moreagents, entities, situations, sets of conditions, etc., that may not beidentical to one another but that are sufficiently similar to permitcomparison there between so that one skilled in the art will appreciatethat conclusions may reasonably be drawn based on differences orsimilarities observed. In some embodiments, comparable sets ofconditions, circumstances, individuals, or populations are characterizedby a plurality of substantially identical features and one or a smallnumber of varied features. Those of ordinary skill in the art willunderstand, in context, what degree of identity is required in any givencircumstance for two or more such agents, entities, situations, sets ofconditions, etc. to be considered comparable. For example, those ofordinary skill in the art will appreciate that sets of circumstances,individuals, or populations are comparable to one another whencharacterized by a sufficient number and type of substantially identicalfeatures to warrant a reasonable conclusion that differences in resultsobtained or phenomena observed under or with different sets ofcircumstances, individuals, or populations are caused by or indicativeof the variation in those features that are varied.

Composition: Those skilled in the art will appreciate that the term“composition” may be used to refer to a discrete physical entity thatcomprises one or more specified components. In general, unless otherwisespecified, a composition may be of any form—e.g., gas, gel, liquid,solid, etc.

Comprising: A composition or method described herein as “comprising” oneor more named elements or steps is open-ended, meaning that the namedelements or steps are essential, but other elements or steps may beadded within the scope of the composition or method. To avoid prolixity,it is also understood that any composition or method described as“comprising” (or which “comprises”) one or more named elements or stepsalso describes the corresponding, more limited composition or method“consisting essentially of” (or which “consists essentially of”) thesame named elements or steps, meaning that the composition or methodincludes the named essential elements or steps and may also includeadditional elements or steps that do not materially affect the basic andnovel characteristic(s) of the composition or method. It is alsounderstood that any composition or method described herein as“comprising” or “consisting essentially of” one or more named elementsor steps also describes the corresponding, more limited, andclosed-ended composition or method “consisting of” (or “consists of”)the named elements or steps to the exclusion of any other unnamedelement or step. In any composition or method disclosed herein, known ordisclosed equivalents of any named essential element or step may besubstituted for that element or step.

Cycloaliphatic: The term “cycloaliphatic,” as used herein, refers tosaturated or partially unsaturated aliphatic monocyclic, bicyclic, orpolycyclic ring systems having, e.g., from 3 to 30, members, wherein thealiphatic ring system is optionally substituted. Cycloaliphatic groupsinclude, without limitation, cyclopropyl, cyclobutyl, cyclopentyl,cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl,cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. Insome embodiments, the cycloalkyl has 3-6 carbons. The terms“cycloaliphatic” may also include aliphatic rings that are fused to oneor more aromatic or nonaromatic rings, such as decahydronaphthyl ortetrahydronaphthyl, where a radical or point of attachment is on analiphatic ring. In some embodiments, a carbocyclic group is bicyclic. Insome embodiments, a carbocyclic group is tricyclic. In some embodiments,a carbocyclic group is polycyclic. In some embodiments, “cycloaliphatic”(or “carbocycle” or “cycloalkyl”) refers to a monocyclic C₃-C₆hydrocarbon, or a C₅-C₁₀ bicyclic hydrocarbon that is completelysaturated or that contains one or more units of unsaturation, but whichis not aromatic, or a C₉-C₁₆ tricyclic hydrocarbon that is completelysaturated or that contains one or more units of unsaturation, but whichis not aromatic.

Derivative: As used herein, the term “derivative” refers to a structuralanalogue of a reference substance. That is, a “derivative” is asubstance that shows significant structural similarity with thereference substance, for example sharing a core or consensus structure,but also differs in certain discrete ways. In some embodiments, aderivative is a substance that can be generated from the referencesubstance by chemical manipulation. In some embodiments, a derivative isa substance that can be generated through performance of a syntheticprocess substantially similar to (e.g., sharing a plurality of stepswith) one that generates the reference substance.

Dosage form or unit dosage form: Those skilled in the art willappreciate that the term “dosage form” may be used to refer to aphysically discrete unit of an active agent (e.g., a therapeutic ordiagnostic agent) for administration to a subject. Typically, each suchunit contains a predetermined quantity of active agent. In someembodiments, such quantity is a unit dosage amount (or a whole fractionthereof) appropriate for administration in accordance with a dosingregimen that has been determined to correlate with a desired orbeneficial outcome when administered to a relevant population (i.e.,with a therapeutic dosing regimen). Those of ordinary skill in the artappreciate that the total amount of a therapeutic composition or agentadministered to a particular subject is determined by one or moreattending physicians and may involve administration of multiple dosageforms.

Dosing regimen: Those skilled in the art will appreciate that the term“dosing regimen” may be used to refer to a set of unit doses (typicallymore than one) that are administered individually to a subject,typically separated by periods of time. In some embodiments, a giventherapeutic agent has a recommended dosing regimen, which may involveone or more doses. In some embodiments, a dosing regimen comprises aplurality of doses each of which is separated in time from other doses.In some embodiments, individual doses are separated from one another bya time period of the same length; in some embodiments, a dosing regimencomprises a plurality of doses and at least two different time periodsseparating individual doses. In some embodiments, all doses within adosing regimen are of the same unit dose amount. In some embodiments,different doses within a dosing regimen are of different amounts. Insome embodiments, a dosing regimen comprises a first dose in a firstdose amount, followed by one or more additional doses in a second doseamount different from the first dose amount. In some embodiments, adosing regimen comprises a first dose in a first dose amount, followedby one or more additional doses in a second dose amount same as thefirst dose amount. In some embodiments, a dosing regimen is correlatedwith a desired or beneficial outcome when administered across a relevantpopulation (i.e., is a therapeutic dosing regimen).

Halogen: The term “halogen” means F, Cl, Br, or I.

Heteroaliphatic: The term “heteroaliphatic” is given its ordinarymeaning in the art and refers to aliphatic groups as described herein inwhich one or more carbon atoms are replaced with one or more heteroatoms(e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, and the like).

Heteroalkyl: The term “heteroalkyl” is given its ordinary meaning in theart and refers to alkyl groups as described herein in which one or morecarbon atoms is replaced with a heteroatom (e.g., oxygen, nitrogen,sulfur, silicon, phosphorus, and the like). Examples of heteroalkylgroups include, but are not limited to, alkoxy, poly(ethylene glycol)-,alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl,etc.

Heteroaryl: The terms “heteroaryl” and “heteroar-,” used alone or aspart of a larger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,”refer to monocyclic, bicyclic or polycyclic ring systems having, forexample, a total of five to thirty, e.g., 5, 6, 9, 10, 14, etc., ringmembers, wherein at least one ring in the system is aromatic and atleast one aromatic ring atom is a heteroatom. In some embodiments, aheteroatom is nitrogen, oxygen or sulfur. In some embodiments, aheteroaryl group is a group having 5 to 10 ring atoms (i.e., monocyclic,bicyclic or polycyclic), in some embodiments 5, 6, 9, or 10 ring atoms.In some embodiments, a heteroaryl group has 6, 10, or 14 π electronsshared in a cyclic array; and having, in addition to carbon atoms, fromone to five heteroatoms. Heteroaryl groups include, without limitation,thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl,tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. In some embodiments, aheteroaryl is a heterobiaryl group, such as bipyridyl and the like. Theterms “heteroaryl” and “heteroar-”, as used herein, also include groupsin which a heteroaromatic ring is fused to one or more aryl,cycloaliphatic, or heterocyclyl rings, where a radical or point ofattachment is on a heteroaromatic ring. Non-limiting examples includeindolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl,indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl,cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl,carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, andpyrido[2,3-b]-1,4-oxazin-3(4H)-one. A heteroaryl group may bemonocyclic, bicyclic or polycyclic. The term “heteroaryl” may be usedinterchangeably with the terms “heteroaryl ring,” “heteroaryl group,” or“heteroaromatic,” any of which terms include rings that are optionallysubstituted. The term “heteroaralkyl” refers to an alkyl groupsubstituted by a heteroaryl group, wherein the alkyl and heteroarylportions independently are optionally substituted.

Heteroatom: The term “heteroatom” means an atom that is not carbon andis not hydrogen. In some embodiments, a heteroatom is oxygen, sulfur,nitrogen, phosphorus, boron or silicon (including any oxidized form ofnitrogen, sulfur, phosphorus, or silicon; the quaternized form of anybasic nitrogen or a substitutable nitrogen of a heterocyclic ring (forexample, N as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl); etc.). In some embodiments, aheteroatom is boron, nitrogen, oxygen, silicon, sulfur, or phosphorus.In some embodiments, a heteroatom is nitrogen, oxygen, silicon, sulfur,or phosphorus. In some embodiments, a heteroatom is nitrogen, oxygen,sulfur, or phosphorus. In some embodiments, a heteroatom is nitrogen,oxygen or sulfur.

Heterocyclyl: As used herein, the terms “heterocycle,” “heterocyclyl,”“heterocyclic radical,” and “heterocyclic ring” are used interchangeablyand refer to a monocyclic, bicyclic or polycyclic ring moiety (e.g.,3-30 membered) that is saturated or partially unsaturated and has one ormore heteroatom ring atoms. In some embodiments, a heteroatom is boron,nitrogen, oxygen, silicon, sulfur, or phosphorus. In some embodiments, aheteroatom is nitrogen, oxygen, silicon, sulfur, or phosphorus. In someembodiments, a heteroatom is nitrogen, oxygen, sulfur, or phosphorus. Insome embodiments, a heteroatom is nitrogen, oxygen or sulfur. In someembodiments, a heterocyclyl group is a stable 5- to 7-memberedmonocyclic or 7- to 10-membered bicyclic heterocyclic moiety that iseither saturated or partially unsaturated, and having, in addition tocarbon atoms, one or more, preferably one to four, heteroatoms, asdefined above. When used in reference to a ring atom of a heterocycle,the term “nitrogen” includes substituted nitrogen. As an example, in asaturated or partially unsaturated ring having 0-3 heteroatoms selectedfrom oxygen, sulfur or nitrogen, the nitrogen may be N (as in3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or ⁺NR (as inN-substituted pyrrolidinyl). A heterocyclic ring can be attached to itspendant group at any heteroatom or carbon atom that results in a stablestructure and any of the ring atoms can be optionally substituted.Examples of such saturated or partially unsaturated heterocyclicradicals include, without limitation, tetrahydrofuranyl,tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl,oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl,thiazepinyl, morpholinyl, and quinuclidinyl. The terms “heterocycle,”“heterocyclyl,” “heterocyclyl ring,” “heterocyclic group,” “heterocyclicmoiety,” and “heterocyclic radical,” are used interchangeably herein,and also include groups in which a heterocyclyl ring is fused to one ormore aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl,3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where aradical or point of attachment is on a heteroaliphatic ring. Aheterocyclyl group may be monocyclic, bicyclic or polycyclic. The term“heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g., between nucleic acidmolecules (e.g., DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% identical. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% similar (e.g., containing residues with relatedchemical properties at corresponding positions). For example, as is wellknown by those of ordinary skill in the art, certain amino acids aretypically classified as similar to one another as “hydrophobic” or“hydrophilic” amino acids, and/or as having “polar” or “non-polar” sidechains. Substitution of one amino acid for another of the same type mayoften be considered a “homologous” substitution. Typical amino acidcategorizations are summarized below:

Alanine Ala A nonpolar neutral 1.8 Arginine Arg R polar positive −4.5Asparagine Asn N polar neutral −3.5 Aspartic acid Asp D polar negative−3.5 Cysteine Cys C nonpolar neutral 2.5 Glutamic acid Glu E polarnegative −3.5 Glutamine Gln Q polar neutral −3.5 Glycine Gly G nonpolarneutral −0.4 Histidine His H polar positive −3.2 Isoleucine Ile Inonpolar neutral 4.5 Leucine Leu L nonpolar neutral 3.8 Lysine Lys Kpolar positive −3.9 Methionine Met M nonpolar neutral 1.9 PhenylalaninePhe F nonpolar neutral 2.8 Proline Pro P nonpolar neutral −1.6 SerineSer S polar neutral −0.8 Threonine Thr T polar neutral −0.7 TryptophanTrp W nonpolar neutral −0.9 Tyrosine Tyr Y polar neutral −1.3 Valine ValV nonpolar neutral 4.2

Ambiguous Amino Acids 3-Letter 1-Letter Asparagine or aspartic acid AsxB Glutamine or glutamic acid Glx Z Leucine or Isoleucine Xle JUnspecified or unknown amino acid Xaa X

As will be understood by those skilled in the art, a variety ofalgorithms are available that permit comparison of sequences in order todetermine their degree of homology, including by permitting gaps ofdesignated length in one sequence relative to another when consideringwhich residues “correspond” to one another in different sequences.Calculation of the percent homology between two nucleic acid sequences,for example, can be performed by aligning the two sequences for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second nucleic acid sequences for optimal alignment andnon-corresponding sequences can be disregarded for comparison purposes).In certain embodiments, the length of a sequence aligned for comparisonpurposes is at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or substantially100% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position; when a position in the first sequence isoccupied by a similar nucleotide as the corresponding position in thesecond sequence, then the molecules are similar at that position. Thepercent homology between the two sequences is a function of the numberof identical and similar positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which needs tobe introduced for optimal alignment of the two sequences. Representativealgorithms and computer programs useful in determining the percenthomology between two nucleotide sequences include, for example, thealgorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has beenincorporated into the ALIGN program (version 2.0) using a PAM120 weightresidue table, a gap length penalty of 12 and a gap penalty of 4. Thepercent homology between two nucleotide sequences can, alternatively, bedetermined for example using the GAP program in the GCG software packageusing an NWSgapdna.CMP matrix.

“Improved,” “increased” or “reduced”: As used herein, these terms, orgrammatically comparable comparative terms, indicate values that arerelative to a comparable reference measurement. For example, in someembodiments, an assessed value achieved with an agent of interest may be“improved” relative to that obtained with a comparable reference agent.Alternatively or additionally, in some embodiments, an assessed valueachieved in a subject or system of interest may be “improved” relativeto that obtained in the same subject or system under differentconditions (e.g., prior to or after an event such as administration ofan agent of interest), or in a different, comparable subject (e.g., in acomparable subject or system that differs from the subject or system ofinterest in presence of one or more indicators of a particular disease,disorder or condition of interest, or in prior exposure to a conditionor agent, etc). In some embodiments, comparative terms refer tostatistically relevant differences (e.g., that are of a prevalenceand/or magnitude sufficient to achieve statistical relevance). Thoseskilled in the art will be aware, or will readily be able to determine,in a given context, a degree and/or prevalence of difference that isrequired or sufficient to achieve such statistical significance.

Partially unsaturated: As used herein, the term “partially unsaturated”refers to a moiety that includes at least one double or triple bond. Theterm “partially unsaturated” is intended to encompass groups havingmultiple sites of unsaturation, but is not intended to include aryl orheteroaryl moieties.

Peptide: The term “peptide” as used herein refers to a polypeptide thatis typically relatively short, for example having a length of less thanabout 100 amino acids, less than about 50 amino acids, less than about40 amino acids less than about 30 amino acids, less than about 25 aminoacids, less than about 20 amino acids, less than about 15 amino acids,or less than 10 amino acids.

Pharmaceutical composition: As used herein, the term “pharmaceuticalcomposition” refers to an active agent, formulated together with one ormore pharmaceutically acceptable carriers. In some embodiments, activeagent is present in unit dose amount appropriate for administration in atherapeutic regimen that shows a statistically significant probabilityof achieving a predetermined therapeutic effect when administered to arelevant population. In some embodiments, pharmaceutical compositionsmay be specially formulated for administration in solid or liquid form,including those adapted for the following: oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),tablets, e.g., those targeted for buccal, sublingual, and systemicabsorption, boluses, powders, granules, pastes for application to thetongue; parenteral administration, for example, by subcutaneous,intramuscular, intravenous or epidural injection as, for example, asterile solution or suspension, or sustained-release formulation;topical application, for example, as a cream, ointment, or acontrolled-release patch or spray applied to the skin, lungs, or oralcavity; intravaginally or intrarectally, for example, as a pessary,cream, or foam; sublingually; ocularly; transdermally; or nasally,pulmonary, and to other mucosal surfaces.

Pharmaceutically acceptable: As used herein, the phrase“pharmaceutically acceptable” refers to those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Pharmaceutically acceptable carrier: As used herein, the term“pharmaceutically acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides;and other non-toxic compatible substances employed in pharmaceuticalformulations.

Pharmaceutically acceptable salt: The term “pharmaceutically acceptablesalt”, as used herein, refers to salts of such compounds that areappropriate for use in pharmaceutical contexts, i.e., salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known. For example, S. M. Berge, et al. describes pharmaceuticallyacceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19(1977). In some embodiments, pharmaceutically acceptable salts include,but are not limited to, nontoxic acid addition salts, which are salts ofan amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, maleic acid, tartaric acid,citric acid, succinic acid or malonic acid or by using other knownmethods such as ion exchange. In some embodiments, pharmaceuticallyacceptable salts include, but are not limited to, adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. In some embodiments, pharmaceutically acceptable saltsinclude, but are not limited to, nontoxic base addition salts, such asthose formed by acidic groups of provided compounds (e.g., phosphatelinkage groups of oligonucleotides, phosphorothioate linkage groups ofoligonucleotides, etc.) with bases. Representative alkali or alkalineearth metal salts include salts of sodium, lithium, potassium, calcium,magnesium, and the like. In some embodiments, pharmaceuticallyacceptable salts are ammonium salts (e.g., —N(R)₃). In some embodiments,pharmaceutically acceptable salts are sodium salts. In some embodiments,pharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and arylsulfonate.

Polypeptide: As used herein refers to any polymeric chain of aminoacids. In some embodiments, a polypeptide has an amino acid sequencethat occurs in nature. In some embodiments, a polypeptide has an aminoacid sequence that does not occur in nature. In some embodiments, apolypeptide has an amino acid sequence that is engineered in that it isdesigned and/or produced through action of the hand of man. In someembodiments, a polypeptide may comprise or consist of natural aminoacids, non-natural amino acids, or both. In some embodiments, apolypeptide may comprise or consist of only natural amino acids or onlynon-natural amino acids. In some embodiments, a polypeptide may compriseD-amino acids, L-amino acids, or both. In some embodiments, apolypeptide may comprise only D-amino acids. In some embodiments, apolypeptide may comprise only L-amino acids. In some embodiments, apolypeptide may include one or more pendant groups or othermodifications, e.g., modifying or attached to one or more amino acidside chains, at the polypeptide's N-terminus, at the polypeptide'sC-terminus, or any combination thereof. In some embodiments, suchpendant groups or modifications may be selected from the groupconsisting of acetylation, amidation, lipidation, methylation,pegylation, etc., including combinations thereof. In some embodiments, apolypeptide may be cyclic, and/or may comprise a cyclic portion. In someembodiments, a polypeptide is not cyclic and/or does not comprise anycyclic portion. In some embodiments, a polypeptide is linear. In someembodiments, a polypeptide may be or comprise a stapled polypeptide. Insome embodiments, the term “polypeptide” may be appended to a name of areference polypeptide, activity, or structure; in such instances it isused herein to refer to polypeptides that share the relevant activity orstructure and thus can be considered to be members of the same class orfamily of polypeptides. For each such class, the present specificationprovides and/or those skilled in the art will be aware of exemplarypolypeptides within the class whose amino acid sequences and/orfunctions are known; in some embodiments, such exemplary polypeptidesare reference polypeptides for the polypeptide class or family. In someembodiments, a member of a polypeptide class or family shows significantsequence homology or identity with, shares a common sequence motif(e.g., a characteristic sequence element) with, and/or shares a commonactivity (in some embodiments at a comparable level or within adesignated range) with a reference polypeptide of the class; in someembodiments with all polypeptides within the class). For example, insome embodiments, a member polypeptide shows an overall degree ofsequence homology or identity with a reference polypeptide that is atleast about 30-40%, and is often greater than about 50%, 60%, 70%, 80%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includesat least one region (e.g., a conserved region that may in someembodiments be or comprise a characteristic sequence element) that showsvery high sequence identity, often greater than 90% or even 95%, 96%,97%, 98%, or 99%. Such a conserved region usually encompasses at least3-4 and often up to 20 or more amino acids; in some embodiments, aconserved region encompasses at least one stretch of at least 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. Insome embodiments, a relevant polypeptide may comprise or consist of afragment of a parent polypeptide. In some embodiments, a usefulpolypeptide as may comprise or consist of a plurality of fragments, eachof which is found in the same parent polypeptide in a different spatialarrangement relative to one another than is found in the polypeptide ofinterest (e.g., fragments that are directly linked in the parent may bespatially separated in the polypeptide of interest or vice versa, and/orfragments may be present in a different order in the polypeptide ofinterest than in the parent), so that the polypeptide of interest is aderivative of its parent polypeptide.

Prevent or prevention: as used herein when used in connection with theoccurrence of a disease, disorder, and/or condition, refers to reducingthe risk of developing the disease, disorder and/or condition and/or todelaying onset of one or more characteristics or symptoms of thedisease, disorder or condition. Prevention may be considered completewhen onset of a disease, disorder or condition has been delayed for apredefined period of time.

Protecting Group: The phrase “protecting group,” as used herein, refersto temporary substituents which protect a potentially reactivefunctional group from undesired chemical transformations. Examples ofsuch protecting groups include esters of carboxylic acids, silyl ethersof alcohols, and acetals and ketals of aldehydes and ketones,respectively. A “Si protecting group” is a protecting group comprising aSi atom, such as Si-trialkyl (e.g., trimethylsilyl, tributylsilyl,t-butyldimethylsilyl), Si-triaryl, Si-alkyl-diphenyl (e.g.,t-butyldiphenylsilyl), or Si-aryl-dialkyl (e.g., Si-phenyldialkyl).Generally, a Si protecting group is attached to an oxygen atom. Thefield of protecting group chemistry has been reviewed (Greene, T. W.;Wuts, P. G. M. Protective Groups in Organic Synthesis, 2nd ed.; Wiley:New York, 1991). Such protecting groups (and associated protectedmoieties) are described in detail below.

Protected hydroxyl groups are well known in the art and include thosedescribed in detail in Protecting Groups in Organic Synthesis, T. W.Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999, theentirety of which is incorporated herein by reference. Examples ofsuitably protected hydroxyl groups further include, but are not limitedto, esters, carbonates, sulfonates, allyl ethers, ethers, silyl ethers,alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers. Examples ofsuitable esters include formates, acetates, propionates, pentanoates,crotonates, and benzoates. Specific examples of suitable esters includeformate, benzoyl formate, chloroacetate, trifluoroacetate,methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate,pivaloate (trimethylacetate), crotonate, 4-methoxy-crotonate, benzoate,p-benzylbenzoate, 2,4,6-trimethylbenzoate. Examples of suitablecarbonates include 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl,2-(trimethylsilyl)ethyl, 2-(phenylsulfonyl)ethyl, vinyl, allyl, andp-nitrobenzyl carbonate. Examples of suitable silyl ethers includetrimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triisopropylsilyl ether, and other trialkylsilylethers. Examples of suitable alkyl ethers include methyl, benzyl,p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and allyl ether,or derivatives thereof. Alkoxyalkyl ethers include acetals such asmethoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl,benzyloxymethyl, beta-(trimethylsilyl)ethoxymethyl, andtetrahydropyran-2-yl ether. Examples of suitable arylalkyl ethersinclude benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl,O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl,p-cyanobenzyl, 2- and 4-picolyl ethers.

Protected amines are well known in the art and include those describedin detail in Greene (1999). Suitable mono-protected amines furtherinclude, but are not limited to, aralkylamines, carbamates, allylamines, amides, and the like. Examples of suitable mono-protected aminomoieties include t-butyloxycarbonylamino (—NHBOC),ethyloxycarbonylamino, methyloxycarbonylamino,trichloroethyloxycarbonylamino, allyloxycarbonylamino (—NHAlloc),benzyloxocarbonylamino (—NHCBZ), allylamino, benzylamino (—NHBn),fluorenylmethylcarbonyl (—NHFmoc), formamido, acetamido,chloroacetamido, dichloroacetamido, trichloroacetamido, phenylacetamido,trifluoroacetamido, benzamido, t-butyldiphenylsilyl, and the like.Suitable di-protected amines include amines that are substituted withtwo substituents independently selected from those described above asmono-protected amines, and further include cyclic imides, such asphthalimide, maleimide, succinimide, and the like. Suitable di-protectedamines also include pyrroles and the like,2,2,5,5-tetramethyl-[1,2,5]azadisilolidine and the like, and azide.

Protected aldehydes are well known in the art and include thosedescribed in detail in Greene (1999). Suitable protected aldehydesfurther include, but are not limited to, acyclic acetals, cyclicacetals, hydrazones, imines, and the like. Examples of such groupsinclude dimethyl acetal, diethyl acetal, diisopropyl acetal, dibenzylacetal, bis(2-nitrobenzyl) acetal, 1,3-dioxanes, 1,3-dioxolanes,semicarbazones, and derivatives thereof.

Protected carboxylic acids are well known in the art and include thosedescribed in detail in Greene (1999). Suitable protected carboxylicacids further include, but are not limited to, optionally substitutedC₁₋₆ aliphatic esters, optionally substituted aryl esters, silyl esters,activated esters, amides, hydrazides, and the like. Examples of suchester groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl,benzyl, and phenyl ester, wherein each group is optionally substituted.Additional suitable protected carboxylic acids include oxazolines andortho esters.

Protected thiols are well known in the art and include those describedin detail in Greene (1999). Suitable protected thiols further include,but are not limited to, disulfides, thioethers, silyl thioethers,thioesters, thiocarbonates, and thiocarbamates, and the like. Examplesof such groups include, but are not limited to, alkyl thioethers, benzyland substituted benzyl thioethers, triphenylmethyl thioethers, andtrichloroethoxycarbonyl thioester, to name but a few.

Reference: As used herein describes a standard or control relative towhich a comparison is performed. For example, in some embodiments, anagent, animal, individual, population, sample, sequence or value ofinterest is compared with a reference or control agent, animal,individual, population, sample, sequence or value. In some embodiments,a reference or control is tested and/or determined substantiallysimultaneously with the testing or determination of interest. In someembodiments, a reference or control is a historical reference orcontrol, optionally embodied in a tangible medium. Typically, as wouldbe understood by those skilled in the art, a reference or control isdetermined or characterized under comparable conditions or circumstancesto those under assessment. Those skilled in the art will appreciate whensufficient similarities are present to justify reliance on and/orcomparison to a particular possible reference or control.

Substitution: As described herein, compounds of the disclosure maycontain optionally substituted and/or substituted moieties. In general,the term “substituted,” whether preceded by the term “optionally” ornot, means that one or more hydrogens of the designated moiety arereplaced with a suitable substituent. Unless otherwise indicated, an“optionally substituted” group may have a suitable substituent at eachsubstitutable position of the group, and when more than one position inany given structure may be substituted with more than one substituentselected from a specified group, the substituent may be either the sameor different at every position. Combinations of substituents envisionedby this disclosure are preferably those that result in the formation ofstable or chemically feasible compounds. The term “stable,” as usedherein, refers to compounds that are not substantially altered whensubjected to conditions to allow for their production, detection, and,in certain embodiments, their recovery, purification, and use for one ormore of the purposes disclosed herein. In some embodiments, examplesubstituents are described below.

Suitable monovalent substituents are halogen; —(CH₂)₀₋₄R^(∘);—(CH₂)₀₋₄OR^(∘); —O(CH₂)₀₋₄R^(∘), —O—(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄CH(OR^(∘))₂; —(CH₂)₀₋₄Ph, which may be substituted with R^(∘);—(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substituted with R^(∘); —CH═CHPh,which may be substituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl whichmay be substituted with R^(∘); —NO₂; —CN; —N₃; —(CH₂)₀₋₄N(R^(∘))₂;—(CH₂)₀₋₄N(R^(∘))C(O)R^(∘); —N(R^(∘))C(S)R^(∘); —(CH₂)₀₋₄N(R^(∘))C(O)N(R^(∘))₂; —N(R^(∘))C(S)N(R^(∘))₂;—(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)N(R^(∘))₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSi(R^(∘))₃; —(CH₂)₀₋₄OC(O)R^(∘);—OC(O)(CH₂)₀₋₄SR^(∘), —SC(S)SR^(∘); —(CH₂)₀₋₄SC(O)R^(∘);—(CH₂)₀₋₄C(O)N(R^(∘))₂; —C(S)N(R^(∘))₂; —C(S)SR^(∘); —SC(S)SR^(∘),—(CH₂)₀₋₄OC(O)N(R^(∘))₂; —C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘);—C(O)CH₂C(O)R^(∘); —C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘);—(CH₂)₀₋₄S(O)₂R^(∘); —(CH₂)₀₋₄S(O)₂₀R^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘);—S(O)₂N(R^(∘))₂; —(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂N(R^(∘))₂;—N(R^(∘))S(O)₂R^(∘); —N(OR^(∘))R^(∘); —C(NH)N(R^(∘))₂; —Si(R^(∘))₃;—OSi(R^(∘))₃; —P(R^(∘))₂; —P(OR^(∘))₂; —OP(R^(∘))₂; —OP(OR^(∘))₂;—N(R^(∘))P(R^(∘))₂; —B(R^(∘))₂; —OB(R^(∘))₂; —P(O)(R^(∘))₂;—OP(O)(R^(∘))₂; —N(R^(∘))P(O)(R^(∘))₂; —(C₁-4 straight or branchedalkylene)O—N(R^(∘))₂; or —(C₁₋₄ straight or branchedalkylene)C(O)O—N(R^(∘))₂; wherein each R^(∘) may be substituted asdefined below and is independently hydrogen, C₁₋₂₀ aliphatic, C₁₋₂₀heteroaliphatic having 1-5 heteroatoms independently selected fromnitrogen, oxygen, sulfur, silicon and phosphorus, —CH₂—(C₆₋₁₄ aryl),—O(CH₂)₀₋₁(C₆₋₁₄ aryl), —CH₂-(5-14 membered heteroaryl ring), a 5-20membered, monocyclic, bicyclic, or polycyclic, saturated, partiallyunsaturated or aryl ring having 0-5 heteroatoms independently selectedfrom nitrogen, oxygen, sulfur, silicon and phosphorus, or,notwithstanding the definition above, two independent occurrences ofR^(∘), taken together with their intervening atom(s), form a 5-20membered, monocyclic, bicyclic, or polycyclic, saturated, partiallyunsaturated or aryl ring having 0-5 heteroatoms independently selectedfrom nitrogen, oxygen, sulfur, silicon and phosphorus, which may besubstituted as defined below.

Suitable monovalent substituents on R^(∘) (or the ring formed by takingtwo independent occurrences of R^(∘) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(●), -(haloR^(●)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(●), —(CH₂)₀₋₂CH(OR^(●))₂; —O(haloR^(●)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(●),—(CH₂)₀₋₂SR^(●), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●),—(CH₂)₀₋₂NR^(●) ₂, —NO₂, —SiR^(●) ₃, —OSiR^(●) ₃, —C(O)SR^(●), —(C₁₋₄straight or branched alkylene)C(O)OR^(●), or —SSR^(●) wherein each R^(●)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur. Suitable divalent substituents on asaturated carbon atom of R^(∘) include ═O and ═S.

Suitable divalent substituents are the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, and sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁-6 aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur.

Suitable substituents on the aliphatic group of R* are halogen, —R^(●),-(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH, —C(O)OR^(●),—NH₂, —NHR^(●), —NR′₂, or —NO₂, wherein each R^(●) is unsubstituted orwhere preceded by “halo” is substituted only with one or more halogens,and is independently C₁-4 aliphatic, —CH₂Ph, —O(CH₂)₀₋₁Ph, or a5-6-membered saturated, partially unsaturated, or aryl ring having 0-4heteroatoms independently selected from nitrogen, oxygen, and sulfur.

In some embodiments, suitable substituents on a substitutable nitrogenare —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†), —C(O)C(O)R^(†),—C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂, —C(S)NR^(†) ₂,—C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R; wherein each R is independentlyhydrogen, C₁₋₆ aliphatic which may be substituted as defined below,unsubstituted —OPh, or an unsubstituted 5-6-membered saturated,partially unsaturated, or aryl ring having 0-4 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur, or, notwithstanding thedefinition above, two independent occurrences of R, taken together withtheir intervening atom(s) form an unsubstituted 3-12-membered saturated,partially unsaturated, or aryl mono- or bicyclic ring having 0-4heteroatoms independently selected from nitrogen, oxygen, and sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN,—C(O)OH, —C(O)OR^(●), —NH₂, —NHR^(●), —NR′₂, or —NO₂, wherein each R^(●)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, and sulfur.

Subject: As used herein, the term “subject” or “test subject” refers toany organism to which a provided compound or composition is administeredin accordance with the present disclosure e.g., for experimental,diagnostic, prophylactic, and/or therapeutic purposes. Typical subjectsinclude animals (e.g., mammals such as mice, rats, rabbits, non-humanprimates, and humans; insects; worms; etc.) and plants. In someembodiments, a subject may be suffering from, and/or susceptible to adisease, disorder, and/or condition. In some embodiments, a subject is ahuman.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition is one who has a higher risk of developingthe disease, disorder, and/or condition than does a member of thegeneral public. In some embodiments, an individual who is susceptible toa disease, disorder and/or condition may not have been diagnosed withthe disease, disorder, and/or condition. In some embodiments, anindividual who is susceptible to a disease, disorder, and/or conditionmay exhibit symptoms of the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,and/or condition may not exhibit symptoms of the disease, disorder,and/or condition. In some embodiments, an individual who is susceptibleto a disease, disorder, and/or condition will develop the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will not developthe disease, disorder, and/or condition.

Therapeutic agent: As used herein, the phrase “therapeutic agent” refersto an agent that, when administered to a subject, has a therapeuticeffect and/or elicits a desired biological and/or pharmacologicaleffect. In some embodiments, a therapeutic agent is any substance thatcan be used to alleviate, ameliorate, relieve, inhibit, prevent, delayonset of, reduce severity of, and/or reduce incidence of one or moresymptoms or features of a disease, disorder, and/or condition.

Therapeutic regimen: A “therapeutic regimen”, as that term is usedherein, refers to a dosing regimen whose administration across arelevant population may be correlated with a desired or beneficialtherapeutic outcome.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of a substance (e.g.,a therapeutic agent, composition, and/or formulation) that elicits adesired biological response when administered as part of a therapeuticregimen. In some embodiments, a therapeutically effective amount of asubstance is an amount that is sufficient, when administered to asubject suffering from or susceptible to a disease, disorder, and/orcondition, to treat, diagnose, prevent, and/or delay the onset of thedisease, disorder, and/or condition. As will be appreciated by those ofordinary skill in this art, the effective amount of a substance may varydepending on such factors as the desired biological endpoint, thesubstance to be delivered, the target cell or tissue, etc. For example,the effective amount of compound in a formulation to treat a disease,disorder, and/or condition is the amount that alleviates, ameliorates,relieves, inhibits, prevents, delays onset of, reduces severity ofand/or reduces incidence of one or more symptoms or features of thedisease, disorder, and/or condition. In some embodiments, atherapeutically effective amount is administered in a single dose; insome embodiments, multiple unit doses are required to deliver atherapeutically effective amount.

Treat: As used herein, the term “treat,” “treatment,” or “treating”refers to any method used to partially or completely alleviate,ameliorate, relieve, inhibit, prevent, delay onset of, reduce severityof, and/or reduce incidence of one or more symptoms or features of adisease, disorder, and/or condition. Treatment may be administered to asubject who does not exhibit signs of a disease, disorder, and/orcondition. In some embodiments, treatment may be administered to asubject who exhibits only early signs of the disease, disorder, and/orcondition, for example for the purpose of decreasing the risk ofdeveloping pathology associated with the disease, disorder, and/orcondition.

Unit dose: The expression “unit dose” as used herein refers to an amountadministered as a single dose and/or in a physically discrete unit of apharmaceutical composition. In many embodiments, a unit dose contains apredetermined quantity of an active agent. In some embodiments, a unitdose contains an entire single dose of the agent. In some embodiments,more than one unit dose is administered to achieve a total single dose.In some embodiments, administration of multiple unit doses is required,or expected to be required, in order to achieve an intended effect. Aunit dose may be, for example, a volume of liquid (e.g., an acceptablecarrier) containing a predetermined quantity of one or more therapeuticagents, a predetermined amount of one or more therapeutic agents insolid form, a sustained release formulation or drug delivery devicecontaining a predetermined amount of one or more therapeutic agents,etc. It will be appreciated that a unit dose may be present in aformulation that includes any of a variety of components in addition tothe therapeutic agent(s). For example, acceptable carriers (e.g.,pharmaceutically acceptable carriers), diluents, stabilizers, buffers,preservatives, etc., may be included as described infra. It will beappreciated by those skilled in the art, in many embodiments, a totalappropriate daily dosage of a particular therapeutic agent may comprisea portion, or a plurality, of unit doses, and may be decided, forexample, by the attending physician within the scope of sound medicaljudgment. In some embodiments, the specific effective dose level for anyparticular subject or organism may depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;activity of specific active compound employed; specific compositionemployed; age, body weight, general health, sex and diet of the subject;time of administration, and rate of excretion of the specific activecompound employed; duration of the treatment; drugs and/or additionaltherapies used in combination or coincidental with specific compound(s)employed, and like factors well known in the medical arts.

Unsaturated: The term “unsaturated” as used herein, means that a moietyhas one or more units of unsaturation.

Wild-type: As used herein, the term “wild-type” has its art-understoodmeaning that refers to an entity having a structure and/or activity asfound in nature in a “normal” (as contrasted with mutant, diseased,altered, etc.) state or context. Those of ordinary skill in the art willappreciate that wild-type genes and polypeptides often exist in multipledifferent forms (e.g., alleles).

Unless otherwise specified, salts, such as pharmaceutically acceptableacid or base addition salts, stereoisomeric forms, and tautomeric forms,of provided compound are included.

Unless otherwise clear from context, in the present disclosure, (i) theterm “a” may be understood to mean “at least one”; (ii) the term “or”may be understood to mean “and/or”; (iii) the terms “comprising” and“including” may be understood to encompass itemized components or stepswhether presented by themselves or together with one or more additionalcomponents or steps; and (iv) the terms “about” and “approximately” maybe understood to permit standard variation as would be understood bythose of ordinary skill in the art; and (v) where ranges are provided,endpoints are included.

2. Beta-Catenin

Beta-catenin is a protein that is important to many biologicalprocesses, e.g., the development of tissue in animals. As part of theWingless and INT-1 (Wnt) signaling pathway, beta-catenin helps toregulate expression of genes, which among other things, are involved incell differentiation, proliferation, and survival. Aberrant Wntsignaling and/or maintenance of beta-catenin levels underlies a numberof human diseases including but not limited to cancer, diabetes, andobesity [Cell. 2012; 149(6): 1192-1205]. It is reported that when Wntsignaling is inactive, beta-catenin resides in a multicomponentdestruction complex that includes the proteins Axin, adenomatouspolypois coli (APC), casein kinase 1α (CK1α), and glycogen synthasekinase 3β (GSK3β). In the destruction complex, beta-catenin may bephosphorylated by CK1α and GSK30. This consequently may tag beta-cateninfor eventual ubiquitination and proteosomal degradation. It is alsoreported that when the Wnt signaling pathway is initiated at thecellular membrane by a ligand, a complex involving the proteins Frizzledand the low-density lipoprotein related receptor (LRP) is formed. Thisheterodimeric protein complex, reportedly, in turn recruits Axin to themembrane resulting in dissociation of the destruction complex andelevated levels of beta-catenin in the cytosol [Dev Cell. 2009; 18(1):9-26].

It is reported that beta-catenin that has accumulated in the cytosol maysubsequently translocate to the nucleus where it may influence theexpression of genes through a transcription activation complex. In somereports, in this complex, beta-catenin associates with several proteinsincluding different transcription factors, histone modifiers, andtranscription co-activators including B-cell CLL/lymphoma 9 (BCL9) [DevCell. 2009; 18(1):9-26]. In some instances, BCL9 serves as a bridgebetween beta-catenin and another protein, pygopus; and studies havedemonstrated that BCL9 mediated recruitment of pygopus is necessary forWnt signaling [Nat Rev Mol Cell Biol. 2009; 10(4): 276-286, Cell. 2002;109(1): 47-60].

In some embodiments, one strategy to address diseases related to Wntsignaling pathway is to affect the ability of β-catenin to interact withother components in the signaling pathway. Reported protein crystalstructures reveal that β-catenin interacts with proteins such as Axinand BCL9 in the destruction complex and transcription complex,respectively. In some reported structures, Axin and BCL9 bind toβ-catenin through interactions mediated by their α-helical Axin-CBD andHD2 domains, respectively. [Genes Dev. 2003; 17(22): 2753-2764, MolCell. 2006; 24(2): 293-300]. While some small molecules have beenreported to modulate β-catenin protein-protein interactions [Curr PharmDes. 2012; 19(40): 634-664], the present disclosure notes that it isgenerally challenging for small molecules to address interaction siteswith extended surface areas as is the case between β-catenin and Axin orBCL9.

In some embodiments, one or more beta-catenin site interacting with Axinare those reported in, e.g., Xing et al., Genes & Development 2003,17(22), 2753-2764. In some embodiments, interactions betweenbeta-catenin and Axin comprise residues 469-481 of Xenopus Axin-CBDdomain (which is highly homologous to human Axin) forming a continuousalpha helix that fits into a groove of beta-catenin formed by thearmadillo repeats. It is reported that Axin-CBD specifically interactswith the third helices of beta-catenin armadillo repeats 3 and 4. Asreported, the beta-catenin/Axin interface is rather hydrophobic.Reported interactions between beta-catenin and Xenopus Axin compriseshydrogen bonding (e.g. side chain of H476 in Xenopus Axin and H260 ofbeta-catenin), salt bridges (e.g. side chain or D474 in Xenopus Axin andK292 of beta-catenin), and/or hydrophobic interaction (e.g. I472, L473,V477, V480, M481 reside on helix surface complementary to a shallowbeta-catenin groove; L473 in Xenopus Axin sits in a shallow hydrophobicpocket formed by F253, F293, and Y254 of beta-catenin; H476 and V477 ofXenopus Axin interact with T257 and I296 of beta-catenin respectively,P469 and M481 of Xenopus Axin interact with S250 and W338 of betacatenin, respectively). In some embodiments, residues 469-481 of XenopusAxin are the human Axin residues corresponding to residues 469-481 inXenopus Axin.

In some embodiments, one or more beta-catenin site interacting with BCL9are those reported in, e.g., Sampietro et al., Molecular Cell, 24(2),293-300, 2006; Kawamoto et al., Biochemistry 2009, 48, 9534-9541; etc.In some embodiments, interactions between beta-catenin and BCL9 comprisethat residues 352-374 of human BCL9-HD2 form a continuous alpha helixthat packs against a groove formed between helices 2 and 3 of armadillorepeat 1 of beta-catenin and forms a helix bundle with the three helicesof the first armadillo repeat of beta-catenin. In some embodiments,interactions between beta-catenin and BCL9 comprises hydrogen bonding,salt bridge, (e.g., the N-terminal side of the BCL9 helix with conservedresidues in beta-catenin that form an acidic knob (e.g., H358 and R359of BCL9 forming hydrogen bond and salt bridge with D162 and D164 ofbeta-catenin, respectively; S362 of BCL9 potentially forming hydrogenbond with H358 of beta-catenin; etc.; see Sampietro 2006)), and/orhydrophobic interaction (e.g., the C-terminal side of BCL9 helix with aconserved beta-catenin surface, involving L366/L369/I373 in BCL9 andresidues L156/L159/L178 of beta-catenin; M174 of beta-catenin protrudinginto hydrophobic interface; etc.; see Sampietro 2006)).

Among other things, the present disclosure provides stapled peptidesthat offer another therapeutic modality for targets such as β-catenin.In some embodiments, compared to small molecules, stapled peptides maybetter address the challenges of targeting protein-protein interactions.In some embodiments, stapled peptides present polypeptide side chainfunctional groups in a desired conformation for competingprotein-protein interactions. Additionally or alternatively, stapledpeptides in some embodiments may possess improved bioactivity,proteolytic stability, and cell permeability, than peptides withoutstaples.

3. Peptide Agents

In some embodiments, provided agents are stapled peptides. In someembodiments, the present disclosure provides stapled peptides thatinteract with beta-catenin. In some embodiments, the present disclosureprovides stapled peptides that interact with beta-catenin and competewith Axin for interaction with beta-catenin. In some embodiments, thepresent disclosure provides stapled peptides that physically interactwith one or more beta-catenin amino acid residues that physicallyinteract with Axin.

Among other things, provided stapled peptides can modulate one or morefunctions of beta-catenin, including those involved in Wnt/beta-cateninpathway. In some embodiments, provided stapled peptides are useful fortreating various conditions, disorders, and/or diseases that areassociated with beta-catenin. Exemplary structural elements of providedstapled peptides are described herein.

a. Amino Acid Sequence

In some embodiments, the present disclosure provides amino acidsequences for stapled peptides. In some embodiments, stapled peptidescomprising provided amino acid sequences interact with beta-catenin,e.g., as determined by one or more methods as described in the presentdisclosure. In some embodiments, stapled peptides comprising providedamino acid sequences interact with beta-catenin at one or morebeta-catenin sites that interact with Axin, e.g., as determined by oneor more methods as described in the present disclosure.

As appreciated by those skilled in the art reading the presentdisclosure, various amino acid sequences, including those specificallyexemplified in the present disclosure and appropriate variants thereof,can be incorporated into provided stapled peptides. In some embodiments,a provided amino acid sequence is derived from a human Axin sequence. Insome embodiments, a provided amino acid sequence is derived from thebeta-catenin binding region of Axin (see Xing, et al.). In someembodiments, a provided amino acid sequence is derived from Axinsequence that interacts with beta-catenin. In some embodiments, aprovided amino acid sequence comprises a sequence of Axin or a variantthereof. In some embodiments, a provided amino acid sequence comprises asequence of the beta-catenin binding region of Axin or a variantthereof. In some embodiments, a provided amino acid sequence comprisesan Axin sequence that interacts with beta-catenin or a variant thereof.In some embodiments, a provided amino acid sequence comprises a set ofAxin residues, or a homolog thereof. In some embodiments, the set ofAxin residues are those that interact with beta-catenin. In someembodiments, the set of Axin residues comprises H476, D474, 1472, L473,V477, V480, P469 and M481 of Xenopus Axin. In some embodiments, the setof Axin residues comprises or is L473, D474, and H476 of Xenopus Axin.In some embodiments, the set of Axin residues comprises H476 of XenopusAxin. In some embodiments, the set of Axin residues comprises D474 ofXenopus Axin. In some embodiments, the set of Axin residues comprisesI472 of Xenopus Axin. In some embodiments, the set of Axin residuescomprises L473 of Xenopus Axin. In some embodiments, the set of Axinresidues comprises V477 of Xenopus Axin. In some embodiments, the set ofAxin residues comprises V480 of Xenopus Axin. In some embodiments, theset of Axin residues comprises P469 of Xenopus Axin. In someembodiments, the set of Axin residues comprises M481 of Xenopus Axin. Insome embodiments, a homolog of a set of Axin residues is a set of Axinresidues wherein one or more amino acid of the set are independentlyreplaced with its or their homologs.

In some embodiments, a homolog of an amino acid is a naturally occurringor non-naturally occurring amino acid that has one or more similarproperties to the amino acid, for example, that is typically classifiedas similar to one another as “hydrophobic”, “hydrophilic”, “basic”, or“acidic” amino acids, and/or as having “polar”, “non-polar”,“hydrophobic”, “hydrophilic”, “basic”, “acidic”, and/or “similar size”side chains. For example, in some embodiments, depending on context, ahomolog of leucine can be an optionally substituted (substituted orunsubstituted) amino acid selected from isoleucine, alanine,homoleucine, 3-cyclobutylalanine, alpha-neopentylglycine,3-cyclopropylalanine, alpha-methylleucine, and 3-cyclohexylalanine; ahomolog of isoleucine can be an optionally substituted amino acidselected from alanine, leucine, homoleucine, 3-cyclobutylalanine,alpha-neopentylglycine, 3-cyclopropylalanine, L-alloisoleucine, andalpha-methylleucine; a homolog of phenylalanine can be an optionallysubstituted amino acid residue selected from tryptophan, tyrosine,3-(1-naphthylalanine), 3-(2-naphthylalanine), 2-chlorophenyalanine,3-chlorophenylalanine, 4-chlorophenylalanine, 4-tert-butylphenylalanine,O-methyl tyrosine, homophenylalanine, 4-fluorophenylalanine,4-methylphenylalanine, 4-bromophenylalanine, 4-phenyl-L-phenylalanine,5-chlorotryptophan, 5-hydroxytryptophan, 4-trifluoromethylphenylalanine,4-guanidino-L-phenylalanine, 2-quinoyl-L-alanine, 3-cyclobutylalanine,alpha-neopentylglycine, and L-2-aminoadipic acid; etc.

In some embodiments, a homolog of a hydrophobic amino acid is anotherhydrophobic amino acid. In some embodiments, a homolog of an amino acidcomprising a hydrophobic side chain is another hydrophobic amino acidcomprising a hydrophobic side chain.

In some embodiments, a homolog of a hydrophilic amino acid is anotherhydrophilic amino acid. In some embodiments, a homolog of an amino acidcomprising a hydrophilic side chain is another hydrophilic amino acidcomprising a hydrophilic side chain.

In some embodiments, a homolog of a basic amino acid is another basicamino acid. In some embodiments, a homolog of an amino acid comprising abasic side chain is another basic amino acid comprising a basic sidechain.

In some embodiments, a homolog of an acidic amino acid is another acidicamino acid. In some embodiments, a homolog of an amino acid comprisingan acidic side chain is another acidic amino acid comprising an acidicside chain.

In some embodiments, a homolog of an aromatic amino acid is anotheraromatic amino acid. In some embodiments, a homolog of an amino acidcomprising an aromatic side chain is another aromatic amino acidcomprising an aromatic side chain.

In some embodiments, a homolog of a polar amino acid is another polaramino acid. In some embodiments, a homolog of an amino acid comprising apolar side chain is another polar amino acid comprising a polar sidechain.

In some embodiments, a homolog of a non-polar amino acid is anothernon-polar amino acid. In some embodiments, a homolog of an amino acidcomprising a non-polar side chain is another non-polar amino acidcomprising a non-polar side chain.

In some embodiments, a homolog of an amino acid is sterically similar tothe amino acid. In some embodiments, a homolog of an amino acidcomprises a side chain that has a similar size to the side chain of theamino acid.

In some embodiments, when an amino acid in a provided agent, e.g., aprovided stapled peptide, is replaced with its homolog, one or moreproperties or activities of the provided agent is not significantlydecreased. For example, in some embodiments, when an amino acid in aprovided stapled peptide is replaced with its homolog, interaction ofthe stapled peptide with beta-catenin is not significantly decreased. Insome embodiments, an interaction is not significantly decreased in thatFP EC50 (e.g., as illustrated in Table 2 measured by competitionfluorescence polarization assay described in the present disclosure(competition with FITC-StAx-33 from Grossmann et al. PNAS 10917942-17947 (FITC-PEG1-PQ-S5-ILD-S5-HVRRVWR (SEQ ID NO: 1), hydrocarbonstaple formed by two S5 via olefin metathesis) orFITC-bA-PQ-S5-ILD-S5-HVRRVWR (SEQ ID NO: 3) (hydrocarbon staple formedby two S5 via olefin metathesis)) after replacement of an amino acidwith its homolog does not increase more than 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000 fold.In some embodiments, an increase is no more than 10 fold. In someembodiments, an increase is no more than 20 fold. In some embodiments,an increase is no more than 30 fold. In some embodiments, an increase isno more than 40 fold. In some embodiments, an increase is no more than50 fold. In some embodiments, an increase is no more than 60 fold. Insome embodiments, an increase is no more than 70 fold. In someembodiments, an increase is no more than 80 fold. In some embodiments,an increase is no more than 90 fold. In some embodiments, an increase isno more than 100 fold. In some embodiments, an increase is no more than200 fold. In some embodiments, an increase is no more than 500 fold. Insome embodiments, as demonstrated in the present disclosure, replacementof an amino acid with a homolog improves one or more properties and/oractivities of a provided stapled peptide. For example, in someembodiments, when an amino acid in a provided stapled peptide isreplaced with its homolog, interaction of the stapled peptide withbeta-catenin is enhanced. In some embodiments, an interaction isenhanced in that FP EC50 (e.g., as illustrated in Table 2 measured bycompetition fluorescence polarization assay described in the presentdisclosure (competition with FITC-StAx-33 from Grossmann et al. PNAS 10917942-17947 (FITC-PEG1-PQ-S5-ILD-S5-HVRRVWR (SEQ ID NO: 1), hydrocarbonstaple formed by two S5 via olefin metathesis) orFITC-bA-PQ-S5-ILD-S5-HVRRVWR (SEQ ID NO: 3) (hydrocarbon staple formedby two S5 via olefin metathesis)) after replacement of an amino acidwith its homolog is decreased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000 fold.In some embodiments, a decrease is at least 2 fold (no more 12 of theoriginal value). In some embodiments, a decrease is at least 3 fold. Insome embodiments, a decrease is at least 4 fold. In some embodiments, adecrease is at least 5 fold. In some embodiments, a decrease is at least6 fold. In some embodiments, a decrease is at least 7 fold. In someembodiments, a decrease is at least 8 fold. In some embodiments, adecrease is at least 9 fold. In some embodiments, a decrease is at least10 fold. In some embodiments, a decrease is at least 15 fold. In someembodiments, a decrease is at least 20 fold. In some embodiments, adecrease is at least 30 fold. In some embodiments, a decrease is atleast 40 fold. In some embodiments, a decrease is at least 50 fold. Insome embodiments, a decrease is at least 60 fold. In some embodiments, adecrease is at least 70 fold. In some embodiments, a decrease is atleast 80 fold. In some embodiments, a decrease is at least 90 fold. Insome embodiments, a decrease is at least 100 fold.

Homologs of amino acids, both naturally occurring and non-naturallyoccurring, may be utilized in amino acid sequences in accordance withthe present disclosure, including those known in the art.

In some embodiments, a homolog of leucine is an optionally substitutedamino acid selected from isoleucine, alanine, homoleucine,3-cyclobutylalanine, alpha-neopentylglycine, and 3-cyclopropylalanine.In some embodiments, a homolog of leucine is isoleucine, alanine,homoleucine, 3-cyclobutylalanine, alpha-neopentylglycine, or3-cyclopropylalanine. In some embodiments, a homolog of leucine is anoptionally substituted amino acid selected from isoleucine,alpha-neopentylglycine, homoleucine, 3-cyclobutylalanine,3-cyclopropylalanine. In some embodiments, a homolog of leucine is anamino acid selected from isoleucine, alpha-neopentylglycine,homoleucine, 3-cyclobutylalanine, 3-cyclopropylalanine. In someembodiments, a homolog of leucine is an optionally substituted aminoacid selected from isoleucine, alpha-neopentylglycine, homoleucine, and3-cyclobutylalanine. In some embodiments, a homolog of leucine is anamino acid selected from isoleucine, alpha-neopentylglycine,homoleucine, and 3-cyclobutylalanine. In some embodiments, a homolog ofleucine is an optionally substituted amino acid selected fromhomoleucine and cyclobutylalanine. In some embodiments, a homolog ofleucine is an amino acid selected from homoleucine andcyclobutylalanine.

In some embodiments, a homolog of isoleucine is an optionallysubstituted amino acid selected from leucine, homoleucine,3-cyclobutylalanine, alpha-neopentylglycine, 3-cyclopropylalanine, andL-alloisoleucine. In some embodiments, a homolog of isoleucine is anamino acid selected from leucine, homoleucine, 3-cyclobutylalanine,alpha-neopentylglycine, 3-cyclopropylalanine, and L-alloisoleucine. Insome embodiments, a homolog of isoleucine is an optionally substitutedamino acid selected from leucine and cyclobutylalanine. In someembodiments, a homolog of isoleucine is an amino acid selected fromleucine and cyclobutylalanine.

In some embodiments, a homolog of phenylalanine is selected from anoptionally substituted amino acid selected from tryptophan,3-(1-naphthylalanine), 3-(2-naphthylalanine), 2-chlorophenyalanine,3-chlorophenylalanine, 4-chlorophenylalanine, 4-tert-butylphenylalanine,O-methyl tyrosine, and homophenylalanine. In some embodiments, a homologof phenylalanine is selected from an amino acid selected fromtryptophan, 3-(1-naphthylalanine), 3-(2-naphthylalanine),2-chlorophenyalanine, 3-chlorophenylalanine, 4-chlorophenylalanine,4-tert-butylphenylalanine, O-methyl tyrosine, and homophenylalanine. Insome embodiments, a homolog of phenylalanine is an optionallysubstituted amino acid selected from 3-(1-naphthylalanine),3-(2-naphthylalanine), 3-chlorophenylalanine, 4-chlorophenylalanine andO-methyl tyrosine. In some embodiments, a homolog of phenylalanine is anamino acid selected from 3-(1-naphthylalanine), 3-(2-naphthylalanine),3-chlorophenylalanine, 4-chlorophenylalanine and O-methyl tyrosine.

In some embodiments, a provided amino acid sequence is or comprises anamino acid sequence or a variant of a peptide selected from Table 1. Insome embodiments, a provided amino acid sequence is or comprises anamino acid sequence or a variant of an amino acid sequence described inSampietro et al., Molecular Cell, 24(2), 293-300, 2006; or Kawamoto etal., Biochemistry 2009, 48, 9534-9541; or WO2017062518; which amino acidsequences are incorporated herein by reference. In some embodiments, aprovided amino acid sequence preferably comprises a set of Axinresidues, or a homolog thereof, as described in the present disclosure.In some embodiments, a provided amino acid sequence comprises one ormore elements reported in the art as required for affinity binding tobeta-catenin, e.g., those reported in Xing, et al.

In some embodiments, a provided amino acid sequence comprises a set ofAxin residues, or a homolog thereof, as described in the presentdisclosure. In some embodiments, a homolog of a set of Axin residues isa set of Axin residues wherein one or more amino acid of the set areindependently replaced with its or their homologs. In some embodiments,a provided amino acid sequence comprises H476, D474, 1472, L473, V477,V480, P469 and M481 of Xenopus Axin, or one or more homologs thereof. Insome embodiments, a provided amino acid sequence comprises L473, D474,and H476 of Xenopus Axin, or one or more homologs thereof. In someembodiments, a provided amino acid sequence comprises H476 of XenopusAxin, or a homolog thereof. In some embodiments, a provided amino acidsequence comprises D474 of Xenopus Axin, or a homolog thereof. In someembodiments, a provided amino acid sequence comprises I472 of XenopusAxin, or a homolog thereof. In some embodiments, a provided amino acidsequence comprises L473 of Xenopus Axin, or a homolog thereof. In someembodiments, a provided amino acid sequence comprises V477 of XenopusAxin, or a homolog thereof. In some embodiments, a provided amino acidsequence comprises V480 of Xenopus Axin, or a homolog thereof. In someembodiments, a provided amino acid sequence comprises P469 of XenopusAxin, or a homolog thereof. In some embodiments, a provided amino acidsequence comprises M481 of Xenopus Axin, or a homolog thereof.

In some embodiments, a provided amino acid sequence comprises H476,D474, I472, L473, V477, V480, P469 and M481 of Xenopus Axin. In someembodiments, a provided amino acid sequence comprises L473, D474, andH476 of Xenopus Axin. In some embodiments, a provided amino acidsequence comprises H476 of Xenopus Axin. In some embodiments, a providedamino acid sequence comprises D474 of Xenopus Axin. In some embodiments,a provided amino acid sequence comprises I472 of Xenopus Axin. In someembodiments, a provided amino acid sequence comprises L473 of XenopusAxin. In some embodiments, a provided amino acid sequence comprises V477of Xenopus Axin. In some embodiments, a provided amino acid sequencecomprises V480 of Xenopus Axin. In some embodiments, a provided aminoacid sequence comprises P469 of Xenopus Axin. In some embodiments, aprovided amino acid sequence comprises M481 of Xenopus Axin.

In some embodiments, a provided amino acid sequence is one that, whenincorporated into a stapled peptide, the stapled peptide interacts withbeta-catenin. In some embodiments, a provided amino acid sequence is onethat, when incorporated into a stapled peptide, the stapled peptideinteracts with beta-catenin and competes with beta-catenin interactionwith Axin. In some embodiments, a provided amino acid sequence is onethat, when incorporated into a stapled peptide, the stapled peptideinteracts with beta-catenin and competes with beta-catenin interactionwith FITC-StAx-33 from Grossmann et al. PNAS 109 17942-17947, and/orFITC-bA-PQ-S5-ILD-S5-HVRRVWR (SEQ ID NO: 3) (hydrocarbon staple formedby two S5 via olefin metathesis). Various assays for assessinginteractions with beta-catenin can be utilized in accordance with thepresent disclosure, including those described in the examples of thepresent disclosure.

In some embodiments, a provided amino acid sequence is homologous to asequence of Axin. In some embodiments, a provided amino acid sequence ishomologous to a sequence of the beta-catenin binding region of Axin. Insome embodiments, a provided amino acid sequence is homologous to asequence of Axin that interacts with beta-catenin. In some embodiments,a provided amino acid sequence is homologous to a sequence of an Axinhelix that interacts with beta-catenin. In some embodiments, a providedamino acid sequence is homologous to a sequence of a peptide describedin Table 1. In some embodiments, a provided amino acid sequence ishomologous to a sequence of a peptide described in Xing et al.

In some embodiments, a provided amino acid sequence is homologous to areference sequence in that the two sequences are at least 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical. In some embodiments, aprovided amino acid sequence is homologous to a reference sequence inthat the two sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% similar (e.g., containing residues with related chemicalproperties at corresponding positions). In some embodiments, tworesidues are considered similar is both of them are hydrophobic,hydrophilic, polar, non-polar, acidic or basic. In some embodiments, tworesidues are considered similar in that one residue is a homolog of theother residue. In some embodiments, a percentage is at least 25%. Insome embodiments, a percentage is at least 30%. In some embodiments, apercentage is at least 35%. In some embodiments, a percentage is atleast 40%. In some embodiments, a percentage is at least 45%. In someembodiments, a percentage is at least 50%. In some embodiments, apercentage is at least 55%. In some embodiments, a percentage is atleast 60%. In some embodiments, a percentage is at least 65%. In someembodiments, a percentage is at least 70%. In some embodiments, apercentage is at least 75%. In some embodiments, a percentage is atleast 80%. In some embodiments, a percentage is at least 85%. In someembodiments, a percentage is at least 90%. In some embodiments, apercentage is at least 91%. In some embodiments, a percentage is atleast 92%. In some embodiments, a percentage is at least 93%. In someembodiments, a percentage is at least 94%. In some embodiments, apercentage is at least 95%. In some embodiments, a percentage is atleast 96%. In some embodiments, a percentage is at least 97%. In someembodiments, a percentage is at least 98%. In some embodiments, apercentage is at least 99%.

Provided amino acid sequences and stapled peptides can be variouslengths, e.g., 2-100, 5-50, 5-40, 5-30, a range from and including 2, 3,4, 5, 6, or 7 to and including 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24 or 25, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 38, 29, or 30 amino acidresidues.

In some embodiments, a length is at least 5 amino acid residues. In someembodiments, a length is at least 6 amino acid residues. In someembodiments, a length is at least 7 amino acid residues. In someembodiments, a length is at least 8 amino acid residues. In someembodiments, a length is at least 9 amino acid residues. In someembodiments, a length is at least 10 amino acid residues. In someembodiments, a length is at least 11 amino acid residues. In someembodiments, a length is at least 12 amino acid residues. In someembodiments, a length is at least 13 amino acid residues. In someembodiments, a length is at least 14 amino acid residues. In someembodiments, a length is at least 15 amino acid residues. In someembodiments, a length is at least 16 amino acid residues. In someembodiments, a length is at least 17 amino acid residues. In someembodiments, a length is at least 18 amino acid residues. In someembodiments, a length is at least 19 amino acid residues. In someembodiments, a length is at least 20 amino acid residues. In someembodiments, a length is at least 21 amino acid residues. In someembodiments, a length is at least 22 amino acid residues. In someembodiments, a length is at least 23 amino acid residues. In someembodiments, a length is at least 24 amino acid residues. In someembodiments, a length is at least 25 amino acid residues.

In some embodiments, a length is 5 amino acid residues. In someembodiments, a length is 6 amino acid residues. In some embodiments, alength is 7 amino acid residues. In some embodiments, a length is 8amino acid residues. In some embodiments, a length is 9 amino acidresidues. In some embodiments, a length is 10 amino acid residues. Insome embodiments, a length is 11 amino acid residues. In someembodiments, a length is 12 amino acid residues. In some embodiments, alength is 13 amino acid residues. In some embodiments, a length is 14amino acid residues. In some embodiments, a length is 15 amino acidresidues. In some embodiments, a length is 16 amino acid residues. Insome embodiments, a length is 17 amino acid residues. In someembodiments, a length is 18 amino acid residues. In some embodiments, alength is 19 amino acid residues. In some embodiments, a length is 20amino acid residues. In some embodiments, a length is 21 amino acidresidues. In some embodiments, a length is 22 amino acid residues. Insome embodiments, a length is 23 amino acid residues. In someembodiments, a length is 24 amino acid residues. In some embodiments, alength is 25 amino acid residues.

In some embodiments, a length is no more than 17 amino acid residues. Insome embodiments, a length is no more than 18 amino acid residues. Insome embodiments, a length is no more than 19 amino acid residues. Insome embodiments, a length is no more than 20 amino acid residues. Insome embodiments, a length is no more than 21 amino acid residues. Insome embodiments, a length is no more than 22 amino acid residues. Insome embodiments, a length is no more than 23 amino acid residues. Insome embodiments, a length is no more than 24 amino acid residues. Insome embodiments, a length is no more than 25 amino acid residues. Insome embodiments, a length is no more than 26 amino acid residues. Insome embodiments, a length is no more than 27 amino acid residues. Insome embodiments, a length is no more than 28 amino acid residues. Insome embodiments, a length is no more than 29 amino acid residues. Insome embodiments, a length is no more than 30 amino acid residues. Insome embodiments, a length is no more than 35 amino acid residues. Insome embodiments, a length is no more than 40 amino acid residues. Insome embodiments, a length is no more than 50 amino acid residues.

Both naturally occurring and non-naturally occurring amino acids can beutilized in accordance with the present disclosure. In some embodiments,an amino acid is a compound comprising an amino group that can form anamide group with a carboxyl group and a carboxyl group.

In some embodiments, an amino acid is a compound having the structure offormula A-I.NH(R^(a1))-L^(a1)-C(R^(a2))(R^(a3))-L^(a2)-COOH,   A-Ior a salt thereof, wherein:

each of Rai, R^(a2), R^(a3) is independently -L^(a)-R′;

each of L^(a), L^(a1) and L^(a2) is independently L;

each L is independently a covalent bond, or an optionally substituted,bivalent C₁-C₂₀ aliphatic group wherein one or more methylene units ofthe aliphatic group are optionally and independently replaced with—C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—,—S(O)₂N(R′)—, —C(O)S—, or —C(O)O—;

each -Cy- is independently an optionally substituted bivalent groupselected from a C₃₋₂₀ cycloaliphatic ring, a C₆₋₂₀ aryl ring, a 5-20membered heteroaryl ring having 1-10 heteroatoms independently selectedfrom oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20membered heterocyclyl ring having 1-10 heteroatoms independentlyselected from oxygen, nitrogen, sulfur, phosphorus and silicon;

each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R;

each R is independently —H, or an optionally substituted group selectedfrom C₁₋₃₀ aliphatic, C₁₋₃₀ heteroaliphatic having 1-10 heteroatomsindependently selected from oxygen, nitrogen, sulfur, phosphorus andsilicon, C₆₋₃₀ aryl, C₆₋₃₀ arylaliphatic, C₆₋₃₀ arylheteroaliphatichaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon, or

two R groups are optionally and independently taken together to form acovalent bond, or:

two or more R groups on the same atom are optionally and independentlytaken together with the atom to form an optionally substituted, 3-30membered, monocyclic, bicyclic or polycyclic ring having, in addition tothe atom, 0-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon; or

two or more R groups on two or more atoms are optionally andindependently taken together with their intervening atoms to form anoptionally substituted, 3-30 membered, monocyclic, bicyclic orpolycyclic ring having, in addition to the intervening atoms, 0-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon.

In some embodiments, L^(a1) is a covalent bond. In some embodiments, acompound of formula A-1 is of the structureNH(R^(a1))—C(R^(a2))(R^(a3))-L^(a2)-COOH.

In some embodiments, L^(a2) is a covalent bond. In some embodiments, acompound of formula A-1 is of the structureNH(R^(a1))—C(R^(a2))(R^(a3))-L^(a2)-COOH.

In some embodiments, L^(a1) is a covalent bond and L^(a2) is a covalentbond. In some embodiments, a compound of formula A-1 is of the structureNH(R^(a1))—C(R^(a))(R^(a3))—COOH.

In some embodiments, L^(a) is a covalent bond. In some embodiments, R′is R. In some embodiments, R^(a1) is R, wherein R is as described in thepresent disclosure. In some embodiments, R^(a2) is R, wherein R is asdescribed in the present disclosure. In some embodiments, R^(a3) is R,wherein R is as described in the present disclosure. In someembodiments, each of R^(a1), R^(a2), and R^(a3) is independently R,wherein R is as described in the present disclosure.

In some embodiments, R^(a1) is hydrogen. In some embodiments, R^(a2) ishydrogen. In some embodiments, R^(a3) is hydrogen. In some embodiments,R^(a1) is hydrogen, and at least one of R^(a2) and R^(a3) is hydrogen.In some embodiments, R^(a1) is hydrogen, one of R^(a2) and R^(a3) ishydrogen, and the other is not hydrogen.

In some embodiments, R^(a2) is -L^(a)-R, wherein R is as described inthe present disclosure. In some embodiments, R^(a2) is -L^(a)-R, whereinR is an optionally substituted group selected from C₃₋₃₀ cycloaliphatic,C₅₋₃₀ aryl, 5-30 membered heteroaryl having 1-10 heteroatomsindependently selected from oxygen, nitrogen, sulfur, phosphorus andsilicon, and 3-30 membered heterocyclyl having 1-10 heteroatomsindependently selected from oxygen, nitrogen, sulfur, phosphorus andsilicon. In some embodiments, R^(a2) is -L^(a)-R, wherein R is anoptionally substituted group selected from C₆₋₃₀ aryl and 5-30 memberedheteroaryl having 1-10 heteroatoms independently selected from oxygen,nitrogen, sulfur, phosphorus and silicon. In some embodiments, R′ is aside chain of an amino acid. In some embodiments, R^(a2) is a side chainof a standard amino acid.

In some embodiments, R^(a3) is -L^(a)-R, wherein R is as described inthe present disclosure. In some embodiments, R^(a3) is -L^(a)-R, whereinR is an optionally substituted group selected from C₃₋₃₀ cycloaliphatic,C₅₋₃₀ aryl, 5-30 membered heteroaryl having 1-10 heteroatomsindependently selected from oxygen, nitrogen, sulfur, phosphorus andsilicon, and 3-30 membered heterocyclyl having 1-10 heteroatomsindependently selected from oxygen, nitrogen, sulfur, phosphorus andsilicon. In some embodiments, R^(a3) is -L^(a)-R, wherein R is anoptionally substituted group selected from C₆₋₃₀ aryl and 5-30 memberedheteroaryl having 1-10 heteroatoms independently selected from oxygen,nitrogen, sulfur, phosphorus and silicon. In some embodiments, R^(a3) isa side chain of an amino acid. In some embodiments, R^(a3) is a sidechain of a standard amino acid.

In some embodiments, in an amino acid, neither of R^(a2) and R^(a3) ishydrogen, e.g., as in certain amino acids exemplified in the presentdisclosure for stapling. In some embodiments, one or both of R^(a2) andR^(a3) comprise an olefin group. An amino acid residue comprising anamino group may form a staple with another amino acid residue comprisingan olefin group through, e.g., olefin metathesis of the olefin groups.In some embodiments, one of R^(a2) and R^(a3) comprises an olefin group.In some embodiments, one of R^(a2) and R^(a3) comprises an olefin group,and the other is optionally substituted C₁₋₄ alkyl. In some embodiments,one of R^(a2) and R^(a3) comprises an olefin group, and the other ismethyl. In some embodiments, both R^(a2) and R^(a3) comprise an olefingroup. In some embodiments, an olefin group is a terminal olefin group.In some embodiments, an olefin group is a terminal olefin group as in anallyl group. In some embodiments, an olefin group is a terminal olefingroup as in an allyloxycarbonyl group. In some embodiments, R^(a2) is analkenyl group comprising a terminal olefin. In some embodiments, R^(a3)is an alkenyl group comprising a terminal olefin. In some embodiments,R^(a2) is —(CH₂)₁₋₁₀—CH═CH₂. In some embodiments, R^(a2) is —CH₂—CH═CH₂.In some embodiments, R^(a2) is —(CH₂)₂—CH═CH₂. In some embodiments,R^(a2) is —(CH₂)₃—CH═CH₂. In some embodiments, R^(a2) is —(CH₂)₄—CH═CH₂.In some embodiments, R^(a2) is —(CH₂)₅—CH═CH₂. In some embodiments,R^(a2) is —(CH₂)₆—CH═CH₂. In some embodiments, R^(a2) is —(CH₂)₇—CH═CH₂.In some embodiments, R^(a2) is —(CH₂)s-CH═CH₂. In some embodiments,R^(a3) is —(CH₂)₁₋₁₀—CH═CH₂. In some embodiments, R^(a3) is —CH₂—CH═CH₂.In some embodiments, R^(a3) is —(CH₂)₂—CH═CH₂. In some embodiments,R^(a3) is —(CH₂)₃—CH═CH₂. In some embodiments, R^(a3) is —(CH₂)₄—CH═CH₂.In some embodiments, R^(a3) is —(CH₂)₅—CH═CH₂. In some embodiments,R^(a3) is —(CH₂)₆—CH═CH₂. In some embodiments, R^(a3) is —(CH₂)₇—CH═CH₂.In some embodiments, R^(a3) is —(CH₂)s-CH═CH₂.

In some embodiments, R^(a2) and R^(a3) are the same. In someembodiments, R^(a2) and R^(a3) are different.

In some embodiments, L^(a) is L, wherein L is as described in thepresent disclosure. In some embodiments, L^(a1) is L, wherein L is asdescribed in the present disclosure. In some embodiments, L^(a2) is L,wherein L is as described in the present disclosure.

In some embodiments, L is a covalent bond, or an optionally substituted,bivalent C₁₋₂₀, e.g., C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂,C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, or C₂₀ aliphatic group wherein one ormore methylene units of the aliphatic group are optionally andindependently replaced with —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, or —C(O)O—. In some embodiments,L is a covalent bond. In some embodiments, L is an optionallysubstituted, bivalent C₁-C₂₀ aliphatic group wherein one or moremethylene units of the aliphatic group are optionally and independentlyreplaced with —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—,—C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—,—S(O)₂N(R′)—, —C(O)S—, or —C(O)O—. In some embodiments, L is anoptionally substituted, bivalent C₁-C₁₅ aliphatic group wherein one ormore methylene units of the aliphatic group are optionally andindependently replaced with —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, or —C(O)O—. In some embodiments,L is an optionally substituted, bivalent C₁-C₁₀ aliphatic group whereinone or more methylene units of the aliphatic group are optionally andindependently replaced with —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, or —C(O)O—.

In some embodiments, at least one methylene group is replaced. In someembodiments, L is an optionally substituted, bivalent C₃-C₂₀ aliphaticgroup wherein one or more methylene units of the aliphatic group areoptionally and independently replaced with —C(R′)₂—, -Cy-, —O—, —S—,—S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, or —C(O)O—. Insome embodiments, L is an optionally substituted, bivalent C₃-C₁₅aliphatic group wherein one or more methylene units of the aliphaticgroup are optionally and independently replaced with —C(R′)₂—, -Cy-,—O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—,or —C(O)O—. In some embodiments, L is an optionally substituted,bivalent C₃-C₁₀ aliphatic group wherein one or more methylene units ofthe aliphatic group are optionally and independently replaced with—C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—,—S(O)₂N(R′)—, —C(O)S—, or —C(O)O—.

In some embodiments, L is an optionally substituted C₁₋₂₀ aliphaticwherein at least one methylene unit is replaced with —N(R′)—. In someembodiments, L is an optionally substituted C₂₋₂₀ aliphatic wherein atleast one methylene unit is replaced with —N(R′)—. In some embodiments,L is an optionally substituted C₃₋₂₀ aliphatic wherein at least onemethylene unit is replaced with —N(R′)—. In some embodiments, L^(a) isL, wherein L is an optionally substituted C₃₋₁₀ aliphatic wherein atleast one methylene unit is replaced with —N(R′)—. In some embodiments,only one methylene unit is replaced with —C(R′)₂—, -Cy-, —O—, —S—,—S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, or —C(O)O—. Insome embodiments, —N(R′)— is —N(O(CO)OR), wherein R is as described inthe present disclosure. In some embodiments, —N(R′)— is -NAlloc-. Insome embodiments, L is optionally substituted C₁₋₆ alkylene. In someembodiments, L is —(CH₂)₂—. In some embodiments, L is —(CH₂)₃—. In someembodiments, L is —(CH₂)₄—. In some embodiments, L is —(CH₂)₅—. In someembodiments, L is —(CH₂)₆—.

In some embodiments, one of R^(2a) and R^(3a) is -L-R′, wherein at leastone methylene unit of L is replaced with —N(R′)—, wherein each of thevariables is independently as described in the present disclosure. Insome embodiments, both the R′ of —N(R′)— and the other of R^(2a) andR^(3a) are R and are taken together with their intervening atoms to forman optionally substituted ring as described in the present disclosure.In some embodiments, a formed ring has no additional heteroatom ringatoms other than the nitrogen atom. In some embodiments, a formed ringis saturated.

In some embodiments, one of R^(2a) and R^(3a) is -L-R′, wherein at leastone methylene unit of L is replaced with —N(R′)C(O)O—, wherein each ofthe variables is independently as described in the present disclosure.In some embodiments, both the R′ of —N(R′)C(O)O— and the other of R^(2a)and R^(3a) are R and are taken together with their intervening atoms toform an optionally substituted ring as described in the presentdisclosure. In some embodiments, a formed ring has no additionalheteroatom ring atoms other than the nitrogen atom. In some embodiments,a formed ring is saturated.

In some embodiments, one of R^(2a) and R^(3a) is —CH₂N(Alloc)CH₃. Insome embodiments, one of R^(2a) and R^(3a) is —(CH₂)₂N(Alloc)CH₃. Insome embodiments, one of R^(2a) and R^(3a) is —(CH₂)₃N(Alloc)CH₃.

In some embodiments, two or more of R^(a1), R^(a2), and R^(a3) are R andare taken together to form an optionally substituted ring as describedin the present disclosure.

In some embodiments, R^(a1) and one of R^(a2) and R^(a3) are R and aretaken together to form an optionally substituted 3-6 membered ringhaving no additional ring heteroatom other than the nitrogen atom towhich R^(a1) is bonded to. In some embodiments, a formed ring is a5-membered ring as in proline.

In some embodiments, R^(a2) and R^(a3) are R and are taken together toform an optionally substituted 3-6 membered ring as described in thepresent disclosure. In some embodiments, R^(a)2 and R^(a3) are R and aretaken together to form an optionally substituted 3-6 membered ringhaving one or more nitrogen ring atom. In some embodiments, R^(a2) andR^(a3) are R and are taken together to form an optionally substituted3-6 membered ring having one and no more than one ring heteroatom whichis a nitrogen atom. In some embodiments, a ring is a saturated ring. Insome embodiments, the nitrogen atom is optionally substituted with analloc group (—N(Alloc)-).

In some embodiments, each -Cy- is independently an optionallysubstituted bivalent group selected from a C₃₋₂₀ cycloaliphatic ring, aC₆₋₂₀ aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatomsindependently selected from oxygen, nitrogen, sulfur, phosphorus andsilicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatomsindependently selected from oxygen, nitrogen, sulfur, phosphorus andsilicon. In some embodiments, -Cy- is an optionally substituted ring asdescribed in the present disclosure, for example, for R and Cy^(L), butis bivalent.

In some embodiments, -Cy- is monocyclic. In some embodiments, -Cy- isbicyclic. In some embodiments, -Cy- is polycyclic. In some embodiments,-Cy- is saturated. In some embodiments, -Cy- is partially unsaturated.In some embodiments, -Cy- is aromatic. In some embodiments, -Cy-comprises a saturated cyclic moiety. In some embodiments, -Cy-comprisesa partially unsaturated cyclic moiety. In some embodiments, -Cy-comprises an aromatic cyclic moiety. In some embodiments, -Cy- comprisesa combination of a saturated, a partially unsaturated, and/or anaromatic cyclic moiety. In some embodiments, -Cy- is 3-membered. In someembodiments, -Cy- is 4-membered. In some embodiments, -Cy- is5-membered. In some embodiments, -Cy- is 6-membered. In someembodiments, -Cy- is 7-membered. In some embodiments, -Cy- is8-membered. In some embodiments, -Cy- is 9-membered. In someembodiments, -Cy- is 10-membered. In some embodiments, -Cy- is11-membered. In some embodiments, -Cy- is 12-membered. In someembodiments, -Cy- is 13-membered. In some embodiments, -Cy- is14-membered. In some embodiments, -Cy- is 15-membered. In someembodiments, -Cy- is 16-membered. In some embodiments, -Cy- is17-membered. In some embodiments, -Cy- is 18-membered. In someembodiments, -Cy- is 19-membered. In some embodiments, -Cy- is20-membered.

In some embodiments, -Cy- is an optionally substituted bivalent C₃₋₂₀cycloaliphatic ring. In some embodiments, -Cy- is an optionallysubstituted bivalent, saturated C₃₋₂₀ cycloaliphatic ring. In someembodiments, -Cy- is an optionally substituted bivalent, partiallyunsaturated C₃₋₂₀ cycloaliphatic ring. In some embodiments, -Cy-H isoptionally substituted cycloaliphatic as described in the presentdisclosure, for example, cycloaliphatic embodiments for R.

In some embodiments, -Cy- is an optionally substituted C₆₋₂₀ aryl ring.In some embodiments, -Cy- is optionally substituted phenylene. In someembodiments, -Cy- is optionally substituted 1,2-phenylene. In someembodiments, -Cy- is optionally substituted 1,3-phenylene. In someembodiments, -Cy- is optionally substituted 1,4-phenylene. In someembodiments, -Cy- is an optionally substituted bivalent naphthalenering. In some embodiments, -Cy-H is optionally substituted aryl asdescribed in the present disclosure, for example, aryl embodiments forR.

In some embodiments, -Cy- is an optionally substituted bivalent 5-20membered heteroaryl ring having 1-10 heteroatoms independently selectedfrom oxygen, nitrogen, sulfur, phosphorus and silicon. In someembodiments, -Cy- is an optionally substituted bivalent 5-20 memberedheteroaryl ring having 1-10 heteroatoms independently selected fromoxygen, nitrogen, and sulfur. In some embodiments, -Cy- is an optionallysubstituted bivalent 5-6 membered heteroaryl ring having 1-4 heteroatomsindependently selected from oxygen, nitrogen, sulfur. In someembodiments, -Cy- is an optionally substituted bivalent 5-6 memberedheteroaryl ring having 1-3 heteroatoms independently selected fromoxygen, nitrogen, sulfur. In some embodiments, -Cy- is an optionallysubstituted bivalent 5-6 membered heteroaryl ring having 1-2 heteroatomsindependently selected from oxygen, nitrogen, sulfur. In someembodiments, -Cy- is an optionally substituted bivalent 5-6 memberedheteroaryl ring having one heteroatom independently selected fromoxygen, nitrogen, sulfur. In some embodiments, -Cy-H is optionallysubstituted heteroaryl as described in the present disclosure, forexample, heteroaryl embodiments for R.

In some embodiments, -Cy- is an optionally substituted bivalent 3-20membered heterocyclyl ring having 1-10 heteroatoms independentlyselected from oxygen, nitrogen, sulfur, phosphorus and silicon. In someembodiments, -Cy- is an optionally substituted bivalent 3-20 memberedheterocyclyl ring having 1-10 heteroatoms independently selected fromoxygen, nitrogen, and sulfur. In some embodiments, -Cy- is an optionallysubstituted bivalent 3-6 membered heterocyclyl ring having 1-4heteroatoms independently selected from oxygen, nitrogen, sulfur. Insome embodiments, -Cy- is an optionally substituted bivalent 5-6membered heterocyclyl ring having 1-4 heteroatoms independently selectedfrom oxygen, nitrogen, sulfur. In some embodiments, -Cy- is anoptionally substituted bivalent 5-6 membered heterocyclyl ring having1-3 heteroatoms independently selected from oxygen, nitrogen, sulfur. Insome embodiments, -Cy- is an optionally substituted bivalent 5-6membered heterocyclyl ring having 1-2 heteroatoms independently selectedfrom oxygen, nitrogen, sulfur. In some embodiments, -Cy- is anoptionally substituted bivalent 5-6 membered heterocyclyl ring havingone heteroatom independently selected from oxygen, nitrogen, sulfur. Insome embodiments, -Cy- is an optionally substituted saturated bivalentheterocyclyl group. In some embodiments, -Cy- is an optionallysubstituted partially unsaturated bivalent heterocyclyl group. In someembodiments, -Cy-H is optionally substituted heterocyclyl as describedin the present disclosure, for example, heterocyclyl embodiments for R.

In some embodiments, R′ is —R, —C(O)R, —C(O)OR, or —S(O)₂R, wherein R isas described in the present disclosure. In some embodiments, R′ is R,wherein R is as described in the present disclosure. In someembodiments, R′ is —C(O)R, wherein R is as described in the presentdisclosure. In some embodiments, R′ is —C(O)OR, wherein R is asdescribed in the present disclosure. In some embodiments, R′ is —S(O)₂R,wherein R is as described in the present disclosure. In someembodiments, R′ is hydrogen. In some embodiments, R′ is not hydrogen. Insome embodiments, R′ is R, wherein R is optionally substituted C₁₋₂₀aliphatic as described in the present disclosure. In some embodiments,R′ is R, wherein R is optionally substituted C₁₋₂₀ heteroaliphatic asdescribed in the present disclosure. In some embodiments, R′ is R,wherein R is optionally substituted C₆₋₂₀ aryl as described in thepresent disclosure. In some embodiments, R′ is R, wherein R isoptionally substituted C₆₋₂₀ arylaliphatic as described in the presentdisclosure. In some embodiments, R′ is R, wherein R is optionallysubstituted C₆₋₂₀ arylheteroaliphatic as described in the presentdisclosure. In some embodiments, R′ is R, wherein R is optionallysubstituted 5-20 membered heteroaryl as described in the presentdisclosure. In some embodiments, R′ is R, wherein R is optionallysubstituted 3-20 membered heterocyclyl as described in the presentdisclosure. In some embodiments, two or more R′ are R, and areoptionally and independently taken together to form an optionallysubstituted ring as described in the present disclosure.

In some embodiments, each R is independently —H, or an optionallysubstituted group selected from C₁₋₃₀ aliphatic, C₁₋₃₀ heteroaliphatichaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon, C₆₋₃₀ aryl, C₆₋₃₀ arylaliphatic, C₆₋₃₀arylheteroaliphatic having 1-10 heteroatoms independently selected fromoxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 memberedheteroaryl having 1-10 heteroatoms independently selected from oxygen,nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclylhaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon, or

two R groups are optionally and independently taken together to form acovalent bond, or:

two or more R groups on the same atom are optionally and independentlytaken together with the atom to form an optionally substituted, 3-30membered, monocyclic, bicyclic or polycyclic ring having, in addition tothe atom, 0-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon; or

two or more R groups on two or more atoms are optionally andindependently taken together with their intervening atoms to form anoptionally substituted, 3-30 membered, monocyclic, bicyclic orpolycyclic ring having, in addition to the intervening atoms, 0-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon.

In some embodiments, each R is independently —H, or an optionallysubstituted group selected from C₁₋₃₀ aliphatic, C₁₋₃₀ heteroaliphatichaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon, C₆₋₃₀ aryl, C₆₋₃₀ arylaliphatic, C₆₋₃₀arylheteroaliphatic having 1-10 heteroatoms independently selected fromoxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 memberedheteroaryl having 1-10 heteroatoms independently selected from oxygen,nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclylhaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon, or

two R groups are optionally and independently taken together to form acovalent bond, or:

two or more R groups on the same atom are optionally and independentlytaken together with the atom to form an optionally substituted, 3-30membered, monocyclic, bicyclic or polycyclic ring having, in addition tothe atom, 0-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon.

two or more R groups on two or more atoms are optionally andindependently taken together with their intervening atoms to form anoptionally substituted, 3-30 membered, monocyclic, bicyclic orpolycyclic ring having, in addition to the intervening atoms, 0-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon.

In some embodiments, each R is independently —H, or an optionallysubstituted group selected from C₁₋₂₀ aliphatic, C₁₋₂₀ heteroaliphatichaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon, C₆₋₂₀ aryl, C₆₋₂₀ arylaliphatic, C₆₋₂₀arylheteroaliphatic having 1-10 heteroatoms independently selected fromoxygen, nitrogen, sulfur, phosphorus and silicon, 5-20 memberedheteroaryl having 1-10 heteroatoms independently selected from oxygen,nitrogen, sulfur, phosphorus and silicon, and 3-20 membered heterocyclylhaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon, or

two R groups are optionally and independently taken together to form acovalent bond, or:

two or more R groups on the same atom are optionally and independentlytaken together with the atom to form an optionally substituted, 3-20membered monocyclic, bicyclic or polycyclic ring having, in addition tothe atom, 0-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon.

two or more R groups on two or more atoms are optionally andindependently taken together with their intervening atoms to form anoptionally substituted, 3-20 membered monocyclic, bicyclic or polycyclicring having, in addition to the intervening atoms, 0-10 heteroatomsindependently selected from oxygen, nitrogen, sulfur, phosphorus andsilicon.

In some embodiments, each R is independently —H, or an optionallysubstituted group selected from C₁₋₃₀ aliphatic, C₁₋₃₀ heteroaliphatichaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon, C₆₋₃₀ aryl, C₆₋₃₀ arylaliphatic, C₆₋₃₀arylheteroaliphatic having 1-10 heteroatoms independently selected fromoxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 memberedheteroaryl having 1-10 heteroatoms independently selected from oxygen,nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclylhaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon.

In some embodiments, each R is independently —H, or an optionallysubstituted group selected from C₁₋₂₀ aliphatic, C₁₋₂₀ heteroaliphatichaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon, C₆₋₂₀ aryl, C₆₋₂₀ arylaliphatic, C₆₋₂₀arylheteroaliphatic having 1-10 heteroatoms independently selected fromoxygen, nitrogen, sulfur, phosphorus and silicon, 5-20 memberedheteroaryl having 1-10 heteroatoms independently selected from oxygen,nitrogen, sulfur, phosphorus and silicon, and 3-20 membered heterocyclylhaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon.

In some embodiments, R is hydrogen. In some embodiments, R is nothydrogen. In some embodiments, R is an optionally substituted groupselected from C₁₋₃₀ aliphatic, C₁₋₃₀ heteroaliphatic having 1-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon, C₆₋₃₀ aryl, a 5-30 membered heteroaryl ringhaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon, and a 3-30 membered heterocyclic ringhaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon.

In some embodiments, R is hydrogen or an optionally substituted groupselected from C₁₋₂₀ aliphatic, phenyl, a 3-7 membered saturated orpartially unsaturated carbocyclic ring, an 8-10 membered bicyclicsaturated, partially unsaturated or aryl ring, a 5-6 membered monocyclicheteroaryl ring having 1-4 heteroatoms independently selected fromnitrogen, oxygen, and sulfur, a 4-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur, a 7-10 membered bicyclicsaturated or partially unsaturated heterocyclic ring having 1-5heteroatoms independently selected from nitrogen, oxygen, and sulfur, oran 8-10 membered bicyclic heteroaryl ring having 1-5 heteroatomsindependently selected from nitrogen, oxygen, and sulfur.

In some embodiments, R is optionally substituted C₁₋₃₀ aliphatic. Insome embodiments, R is optionally substituted C₁₋₂₀ aliphatic. In someembodiments, R is optionally substituted C₁₋₁₅ aliphatic. In someembodiments, R is optionally substituted C₁₋₁₀ aliphatic. In someembodiments, R is optionally substituted C₁₋₆ aliphatic. In someembodiments, R is optionally substituted C₁₋₆ alkyl. In someembodiments, R is optionally substituted hexyl, pentyl, butyl, propyl,ethyl or methyl. In some embodiments, R is optionally substituted hexyl.In some embodiments, R is optionally substituted pentyl. In someembodiments, R is optionally substituted butyl. In some embodiments, Ris optionally substituted propyl. In some embodiments, R is optionallysubstituted ethyl. In some embodiments, R is optionally substitutedmethyl. In some embodiments, R is hexyl. In some embodiments, R ispentyl. In some embodiments, R is butyl. In some embodiments, R ispropyl. In some embodiments, R is ethyl. In some embodiments, R ismethyl. In some embodiments, R is isopropyl. In some embodiments, R isn-propyl. In some embodiments, R is tert-butyl. In some embodiments, Ris sec-butyl. In some embodiments, R is n-butyl. In some embodiments, Ris —(CH₂)₂CN.

In some embodiments, R is optionally substituted C₃₋₃₀ cycloaliphatic.In some embodiments, R is optionally substituted C₃₋₂₀ cycloaliphatic.In some embodiments, R is optionally substituted C₃₋₁₀ cycloaliphatic.In some embodiments, R is optionally substituted cyclohexyl. In someembodiments, R is cyclohexyl. In some embodiments, R is optionallysubstituted cyclopentyl. In some embodiments, R is cyclopentyl. In someembodiments, R is optionally substituted cyclobutyl. In someembodiments, R is cyclobutyl. In some embodiments, R is optionallysubstituted cyclopropyl. In some embodiments, R is cyclopropyl.

In some embodiments, R is an optionally substituted 3-30 memberedsaturated or partially unsaturated carbocyclic ring. In someembodiments, R is an optionally substituted 3-7 membered saturated orpartially unsaturated carbocyclic ring. In some embodiments, R is anoptionally substituted 3-membered saturated or partially unsaturatedcarbocyclic ring. In some embodiments, R is an optionally substituted4-membered saturated or partially unsaturated carbocyclic ring. In someembodiments, R is an optionally substituted 5-membered saturated orpartially unsaturated carbocyclic ring. In some embodiments, R is anoptionally substituted 6-membered saturated or partially unsaturatedcarbocyclic ring. In some embodiments, R is an optionally substituted7-membered saturated or partially unsaturated carbocyclic ring. In someembodiments, R is optionally substituted cycloheptyl. In someembodiments, R is cycloheptyl. In some embodiments, R is optionallysubstituted cyclohexyl. In some embodiments, R is cyclohexyl. In someembodiments, R is optionally substituted cyclopentyl. In someembodiments, R is cyclopentyl. In some embodiments, R is optionallysubstituted cyclobutyl. In some embodiments, R is cyclobutyl. In someembodiments, R is optionally substituted cyclopropyl. In someembodiments, R is cyclopropyl.

In some embodiments, when R is or comprises a ring structure, e.g.,cycloaliphatic, cycloheteroaliphatic, aryl, heteroaryl, etc., the ringstructure can be monocyclic, bicyclic or polycyclic. In someembodiments, R is or comprises a monocyclic structure. In someembodiments, R is or comprises a bicyclic structure. In someembodiments, R is or comprises a polycyclic structure.

In some embodiments, R is optionally substituted C₁₋₃₀ heteroaliphatichaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon. In some embodiments, R is optionallysubstituted C₁₋₂₀ heteroaliphatic having 1-10 heteroatoms. In someembodiments, R is optionally substituted C₁₋₂₀ heteroaliphatic having1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus or silicon, optionally including one or more oxidized formsof nitrogen, sulfur, phosphorus or selenium. In some embodiments, R isoptionally substituted C₁₋₃₀ heteroaliphatic comprising 1-10 groupsindependently selected from

In some embodiments, R is optionally substituted C₆₋₃₀ aryl. In someembodiments, R is optionally substituted phenyl. In some embodiments, Ris phenyl. In some embodiments, R is substituted phenyl.

In some embodiments, R is an optionally substituted 8-10 memberedbicyclic saturated, partially unsaturated or aryl ring. In someembodiments, R is an optionally substituted 8-10 membered bicyclicsaturated ring. In some embodiments, R is an optionally substituted 8-10membered bicyclic partially unsaturated ring. In some embodiments, R isan optionally substituted 8-10 membered bicyclic aryl ring. In someembodiments, R is optionally substituted naphthyl.

In some embodiments, R is optionally substituted 5-30 memberedheteroaryl ring having 1-10 heteroatoms independently selected fromoxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, Ris optionally substituted 5-30 membered heteroaryl ring having 1-10heteroatoms independently selected from oxygen, nitrogen, and sulfur. Insome embodiments, R is optionally substituted 5-30 membered heteroarylring having 1-5 heteroatoms independently selected from oxygen,nitrogen, sulfur, phosphorus and silicon. In some embodiments, R isoptionally substituted 5-30 membered heteroaryl ring having 1-5heteroatoms independently selected from oxygen, nitrogen, and sulfur.

In some embodiments, R is an optionally substituted 5-6 memberedmonocyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur. In some embodiments, R is asubstituted 5-6 membered monocyclic heteroaryl ring having 1-4heteroatoms independently selected from nitrogen, oxygen, and sulfur. Insome embodiments, R is an unsubstituted 5-6 membered monocyclicheteroaryl ring having 1-4 heteroatoms independently selected fromnitrogen, oxygen, and sulfur. In some embodiments, R is an optionallysubstituted 5-6 membered monocyclic heteroaryl ring having 1-3heteroatoms independently selected from nitrogen, sulfur, and oxygen. Insome embodiments, R is a substituted 5-6 membered monocyclic heteroarylring having 1-3 heteroatoms independently selected from nitrogen,oxygen, and sulfur. In some embodiments, R is an unsubstituted 5-6membered monocyclic heteroaryl ring having 1-3 heteroatoms independentlyselected from nitrogen, sulfur, and oxygen.

In some embodiments, R is an optionally substituted 5-memberedmonocyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen or sulfur. In some embodiments, R is an optionallysubstituted 6-membered monocyclic heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, and sulfur.

In some embodiments, R is an optionally substituted 5-memberedmonocyclic heteroaryl ring having one heteroatom selected from nitrogen,oxygen, and sulfur. In some embodiments, R is selected from optionallysubstituted pyrrolyl, furanyl, or thienyl.

In some embodiments, R is an optionally substituted 5-memberedheteroaryl ring having two heteroatoms independently selected fromnitrogen, oxygen, and sulfur. In certain embodiments, R is an optionallysubstituted 5-membered heteroaryl ring having one nitrogen atom, and anadditional heteroatom selected from sulfur or oxygen. Example R groupsinclude but are not limited to optionally substituted pyrazolyl,imidazolyl, thiazolyl, isothiazolyl, oxazolyl or isoxazolyl.

In some embodiments, R is an optionally substituted 5-memberedheteroaryl ring having three heteroatoms independently selected fromnitrogen, oxygen, and sulfur. Example R groups include but are notlimited to optionally substituted triazolyl, oxadiazolyl orthiadiazolyl.

In some embodiments, R is an optionally substituted 5-memberedheteroaryl ring having four heteroatoms independently selected fromnitrogen, oxygen, and sulfur. Example R groups include but are notlimited to optionally substituted tetrazolyl, oxatriazolyl andthiatriazolyl.

In some embodiments, R is an optionally substituted 6-memberedheteroaryl ring having 1-4 nitrogen atoms. In some embodiments, R is anoptionally substituted 6-membered heteroaryl ring having 1-3 nitrogenatoms. In other embodiments, R is an optionally substituted 6-memberedheteroaryl ring having 1-2 nitrogen atoms. In some embodiments, R is anoptionally substituted 6-membered heteroaryl ring having four nitrogenatoms. In some embodiments, R is an optionally substituted 6-memberedheteroaryl ring having three nitrogen atoms. In some embodiments, R isan optionally substituted 6-membered heteroaryl ring having two nitrogenatoms. In certain embodiments, R is an optionally substituted 6-memberedheteroaryl ring having one nitrogen atom. Example R groups include butare not limited to optionally substituted pyridinyl, pyrimidinyl,pyrazinyl, pyridazinyl, triazinyl, or tetrazinyl.

In certain embodiments, R is an optionally substituted 8-10 memberedbicyclic heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur. In some embodiments, R is anoptionally substituted 5,6-fused heteroaryl ring having 1-4 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In otherembodiments, R is an optionally substituted 5,6-fused heteroaryl ringhaving 1-2 heteroatoms independently selected from nitrogen, oxygen, andsulfur. In certain embodiments, R is an optionally substituted 5,6-fusedheteroaryl ring having 1 heteroatom independently selected fromnitrogen, oxygen, and sulfur. In some embodiments, R is an optionallysubstituted indolyl. In some embodiments, R is an optionally substitutedazabicyclo[3.2.1]octanyl. In certain embodiments, R is an optionallysubstituted 5,6-fused heteroaryl ring having 2 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur. In some embodiments, R is anoptionally substituted azaindolyl. In some embodiments, R is anoptionally substituted benzimidazolyl. In some embodiments, R is anoptionally substituted benzothiazolyl. In some embodiments, R is anoptionally substituted benzoxazolyl. In some embodiments, R is anoptionally substituted indazolyl. In certain embodiments, R is anoptionally substituted 5,6-fused heteroaryl ring having 3 heteroatomsindependently selected from nitrogen, oxygen, and sulfur.

In some embodiments, R is an optionally substituted 5,6-fused heteroarylring having 1-5 heteroatoms independently selected from nitrogen,oxygen, and sulfur. In some embodiments, R is an optionally substituted5,6-fused heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur. In some embodiments, R is anoptionally substituted 5,6-fused heteroaryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, R is an optionally substituted 5,6-fused heteroaryl ringhaving two heteroatoms independently selected from nitrogen, oxygen, andsulfur. In some embodiments, R is an optionally substituted 5,6-fusedheteroaryl ring having three heteroatoms independently selected fromnitrogen, oxygen, and sulfur. In some embodiments, R is an optionallysubstituted 5,6-fused heteroaryl ring having four heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, R is an optionally substituted 5,6-fused heteroaryl ringhaving five heteroatoms independently selected from nitrogen, oxygen,and sulfur.

In certain embodiments, R is an optionally substituted 5,6-fusedheteroaryl ring having one heteroatom independently selected fromnitrogen, oxygen, and sulfur. In some embodiments, R is optionallysubstituted indolyl. In some embodiments, R is optionally substitutedbenzofuranyl. In some embodiments, R is optionally substitutedbenzo[b]thienyl. In certain embodiments, R is an optionally substituted5,6-fused heteroaryl ring having two heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur. In some embodiments, R is optionallysubstituted azaindolyl. In some embodiments, R is optionally substitutedbenzimidazolyl. In some embodiments, R is optionally substitutedbenzothiazolyl. In some embodiments, R is optionally substitutedbenzoxazolyl. In some embodiments, R is an optionally substitutedindazolyl. In certain embodiments, R is an optionally substituted5,6-fused heteroaryl ring having three heteroatoms independentlyselected from nitrogen, oxygen, and sulfur. In some embodiments, R isoptionally substituted oxazolopyridiyl, thiazolopyridinyl orimidazopyridinyl. In certain embodiments, R is an optionally substituted5,6-fused heteroaryl ring having four heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur. In some embodiments, R is optionallysubstituted purinyl, oxazolopyrimidinyl, thiazolopyrimidinyl,oxazolopyrazinyl, thiazolopyrazinyl, imidazopyrazinyl,oxazolopyridazinyl, thiazolopyridazinyl or imidazopyridazinyl. Incertain embodiments, R is an optionally substituted 5,6-fused heteroarylring having five heteroatoms independently selected from nitrogen,oxygen, and sulfur.

In some embodiments, R is optionally substituted1,4-dihydropyrrolo[3,2-b]pyrrolyl, 4H-furo[3,2-b]pyrrolyl,4H-thieno[3,2-b]pyrrolyl, furo[3,2-b]furanyl, thieno[3,2-b]furanyl,thieno[3,2-b]thienyl, 1H-pyrrolo[1,2-a]imidazolyl,pyrrolo[2,1-b]oxazolyl or pyrrolo[2,1-b]thiazolyl. In some embodiments,R is optionally substituted dihydropyrroloimidazolyl, 1H-furoimidazolyl,1H-thienoimidazolyl, furooxazolyl, furoisoxazolyl, 4H-pyrrolooxazolyl,4H-pyrroloisoxazolyl, thienooxazolyl, thienoisoxazolyl,4H-pyrrolothiazolyl, furothiazolyl, thienothiazolyl,1H-imidazoimidazolyl, imidazooxazolyl or imidazo[5,1-b]thiazolyl.

In certain embodiments, R is an optionally substituted 6,6-fusedheteroaryl ring having 1-4 heteroatoms independently selected fromnitrogen, oxygen, and sulfur. In some embodiments, R is an optionallysubstituted 6,6-fused heteroaryl ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In otherembodiments, R is an optionally substituted 6,6-fused heteroaryl ringhaving 1 heteroatom independently selected from nitrogen, oxygen, andsulfur. In some embodiments, R is an optionally substituted quinolinyl.In some embodiments, R is an optionally substituted isoquinolinyl. Insome embodiments, R is an optionally substituted 6,6-fused heteroarylring having 2 heteroatoms independently selected from nitrogen, oxygen,and sulfur. In some embodiments, R is optionally substituted quinazolineor a quinoxaline.

In some embodiments, R is 3-30 membered heterocyclic ring having 1-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon. In some embodiments, R is 3-30 memberedheterocyclic ring having 1-10 heteroatoms independently selected fromoxygen, nitrogen, and sulfur. In some embodiments, R is 3-30 memberedheterocyclic ring having 1-5 heteroatoms independently selected fromoxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, Ris 3-30 membered heterocyclic ring having 1-5 heteroatoms independentlyselected from oxygen, nitrogen, and sulfur.

In some embodiments, R is an optionally substituted 3-7 memberedsaturated or partially unsaturated heterocyclic ring having 1-3heteroatoms independently selected from nitrogen, oxygen, and sulfur. Insome embodiments, R is a substituted 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur. In some embodiments, R is anunsubstituted 3-7 membered saturated or partially unsaturatedheterocyclic ring having 1-3 heteroatoms independently selected fromnitrogen, oxygen, and sulfur. In certain embodiments, R is an optionallysubstituted 5-7 membered partially unsaturated monocyclic ring having1-3 heteroatoms independently selected from nitrogen, oxygen, andsulfur. In certain embodiments, R is an optionally substituted 5-6membered partially unsaturated monocyclic ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In certainembodiments, R is an optionally substituted 5-membered partiallyunsaturated monocyclic ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur. In certain embodiments, R isan optionally substituted 6-membered partially unsaturated monocyclicring having 1-3 heteroatoms independently selected from nitrogen,oxygen, and sulfur. In certain embodiments, R is an optionallysubstituted 7-membered partially unsaturated monocyclic ring having 1-3heteroatoms independently selected from nitrogen, oxygen, and sulfur. Insome embodiments, R is optionally substituted 3-membered heterocyclicring having one heteroatom selected from nitrogen, oxygen or sulfur. Insome embodiments, R is optionally substituted 4-membered heterocyclicring having 1-3 heteroatoms independently selected from nitrogen,oxygen, and sulfur. In some embodiments, R is optionally substituted5-membered heterocyclic ring having 1-3 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur. In some embodiments, R isoptionally substituted 6-membered heterocyclic ring having 1-3heteroatoms independently selected from nitrogen, oxygen, and sulfur. Insome embodiments, R is optionally substituted 7-membered heterocyclicring having 1-3 heteroatoms independently selected from nitrogen,oxygen, and sulfur.

In some embodiments, R is an optionally substituted 3-membered saturatedor partially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, R is an optionally substituted 4-membered saturated orpartially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, R is an optionally substituted 5-membered saturated orpartially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, R is an optionally substituted 6-membered saturated orpartially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, R is an optionally substituted 7-membered saturated orpartially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, and sulfur.

In some embodiments, R is an optionally substituted 4-membered saturatedor partially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, R is an optionally substituted 4-membered partiallyunsaturated heterocyclic ring having 2 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur. In some embodiments, R is anoptionally substituted 4-membered partially unsaturated heterocyclicring having no more than 1 heteroatom. In some embodiments, R is anoptionally substituted 4-membered partially unsaturated heterocyclicring having no more than 1 heteroatom, wherein the heteroatom isnitrogen. In some embodiments, R is an optionally substituted 4-memberedpartially unsaturated heterocyclic ring having no more than 1heteroatom, wherein the heteroatom is oxygen. In some embodiments, R isan optionally substituted 4-membered partially unsaturated heterocyclicring having no more than 1 heteroatom, wherein the heteroatom is sulfur.In some embodiments, R is an optionally substituted 4-membered partiallyunsaturated heterocyclic ring having 2 oxygen atoms. In someembodiments, R is an optionally substituted 4-membered partiallyunsaturated heterocyclic ring having 2 nitrogen atoms. In someembodiments, R is an optionally substituted 4-membered saturated orpartially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, R is an optionally substituted 4-membered partiallyunsaturated heterocyclic ring having 2 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur. In some embodiments, R is anoptionally substituted 4-membered partially unsaturated heterocyclicring having no more than 1 heteroatom. In some embodiments, R is anoptionally substituted 4-membered partially unsaturated heterocyclicring having no more than 1 heteroatom, wherein the heteroatom isnitrogen. In some embodiments, R is an optionally substituted 4-memberedpartially unsaturated heterocyclic ring having no more than 1heteroatom, wherein the heteroatom is oxygen. In some embodiments, R isan optionally substituted 4-membered partially unsaturated heterocyclicring having no more than 1 heteroatom, wherein the heteroatom is sulfur.In some embodiments, R is an optionally substituted 4-membered partiallyunsaturated heterocyclic ring having 2 oxygen atoms. In someembodiments, R is an optionally substituted 4-membered partiallyunsaturated heterocyclic ring having 2 nitrogen atoms.

In some embodiments, R is an optionally substituted 5-membered saturatedor partially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, R is an optionally substituted 5-membered partiallyunsaturated heterocyclic ring having 2 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur. In some embodiments, R is anoptionally substituted 5-membered partially unsaturated heterocyclicring having no more than 1 heteroatom. In some embodiments, R is anoptionally substituted 5-membered partially unsaturated heterocyclicring having no more than 1 heteroatom, wherein the heteroatom isnitrogen. In some embodiments, R is an optionally substituted 5-memberedpartially unsaturated heterocyclic ring having no more than 1heteroatom, wherein the heteroatom is oxygen. In some embodiments, R isan optionally substituted 5-membered partially unsaturated heterocyclicring having no more than 1 heteroatom, wherein the heteroatom is sulfur.In some embodiments, R is an optionally substituted 5-membered partiallyunsaturated heterocyclic ring having 2 oxygen atoms. In someembodiments, R is an optionally substituted 5-membered partiallyunsaturated heterocyclic ring having 2 nitrogen atoms.

In some embodiments, R is an optionally substituted 6-membered saturatedor partially unsaturated heterocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, R is an optionally substituted 6-membered partiallyunsaturated heterocyclic ring having 2 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur. In some embodiments, R is anoptionally substituted 6-membered partially unsaturated heterocyclicring having no more than 1 heteroatom. In some embodiments, R is anoptionally substituted 6-membered partially unsaturated heterocyclicring having no more than 1 heteroatom, wherein the heteroatom isnitrogen. In some embodiments, R is an optionally substituted 6-memberedpartially unsaturated heterocyclic ring having no more than 1heteroatom, wherein the heteroatom is oxygen. In some embodiments, R isan optionally substituted 6-membered partially unsaturated heterocyclicring having no more than 1 heteroatom, wherein the heteroatom is sulfur.In some embodiments, R is an optionally substituted 6-membered partiallyunsaturated heterocyclic ring having 2 oxygen atoms. In someembodiments, R is an optionally substituted 6-membered partiallyunsaturated heterocyclic ring having 2 nitrogen atoms.

In certain embodiments, R is a 3-7 membered saturated or partiallyunsaturated heterocyclic ring having 1-2 heteroatoms independentlyselected from nitrogen, oxygen, and sulfur. In certain embodiments, R isoptionally substituted oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, oxepaneyl, aziridineyl, azetidineyl, pyrrolidinyl,piperidinyl, azepanyl, thiiranyl, thietanyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, thiepanyl, dioxolanyl, oxathiolanyl,oxazolidinyl, imidazolidinyl, thiazolidinyl, dithiolanyl, dioxanyl,morpholinyl, oxathianyl, piperazinyl, thiomorpholinyl, dithianyl,dioxepanyl, oxazepanyl, oxathiepanyl, dithiepanyl, diazepanyl,dihydrofuranonyl, tetrahydropyranonyl, oxepanonyl, pyrolidinonyl,piperidinonyl, azepanonyl, dihydrothiophenonyl, tetrahydrothiopyranonyl,thiepanonyl, oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl,dioxanonyl, dioxepanonyl, oxathiolinonyl, oxathianonyl, oxathiepanonyl,thiazolidinonyl, thiazinanonyl, thiazepanonyl, imidazolidinonyl,tetrahydropyrimidinonyl, diazepanonyl, imidazolidinedionyl,oxazolidinedionyl, thiazolidinedionyl, dioxolanedionyl,oxathiolanedionyl, piperazinedionyl, morpholinedionyl,thiomorpholinedionyl, tetrahydropyranyl, tetrahydrofuranyl, morpholinyl,thiomorpholinyl, piperidinyl, piperazinyl, pyrrolidinyl,tetrahydrothiophenyl, or tetrahydrothiopyranyl.

In certain embodiments, R is an optionally substituted 5-6 memberedpartially unsaturated monocyclic ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In certainembodiments, R is an optionally substituted tetrahydropyridinyl,dihydrothiazolyl, dihydrooxazolyl, or oxazolinyl group.

In some embodiments, R is an optionally substituted 7-10 memberedbicyclic saturated or partially unsaturated heterocyclic ring having 1-5heteroatoms independently selected from nitrogen, oxygen, and sulfur. Insome embodiments, R is optionally substituted indolinyl. In someembodiments, R is optionally substituted isoindolinyl. In someembodiments, R is optionally substituted 1, 2, 3,4-tetrahydroquinolinyl. In some embodiments, R is optionally substituted1, 2, 3, 4-tetrahydroisoquinolinyl. In some embodiments, R is anoptionally substituted azabicyclo[3.2.1]octanyl.

In some embodiments, R is an optionally substituted 8-10 memberedbicyclic heteroaryl ring having 1-5 heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur.

In some embodiments, R is an optionally substituted 5,6-fused heteroarylring having 1-5 heteroatoms independently selected from nitrogen,oxygen, and sulfur. In some embodiments, R is an optionally substituted5,6-fused heteroaryl ring having 1-4 heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur. In some embodiments, R is anoptionally substituted 5,6-fused heteroaryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, R is an optionally substituted 5,6-fused heteroaryl ringhaving two heteroatoms independently selected from nitrogen, oxygen, andsulfur. In some embodiments, R is optionally substituted1,4-dihydropyrrolo[3,2-b]pyrrolyl, 4H-furo[3,2-b]pyrrolyl,4H-thieno[3,2-b]pyrrolyl, furo[3,2-b]furanyl, thieno[3,2-b]furanyl,thieno[3,2-b]thienyl, 1H-pyrrolo[1,2-a]imidazolyl,pyrrolo[2,1-b]oxazolyl or pyrrolo[2,1-b]thiazolyl. In some embodiments,R is an optionally substituted 5,6-fused heteroaryl ring having threeheteroatoms independently selected from nitrogen, oxygen, and sulfur. Insome embodiments, R is optionally substituted dihydropyrroloimidazolyl,1H-furoimidazolyl, 1H-thienoimidazolyl, furooxazolyl, furoisoxazolyl,4H-pyrrolooxazolyl, 4H-pyrroloisoxazolyl, thienooxazolyl,thienoisoxazolyl, 4H-pyrrolothiazolyl, furothiazolyl, thienothiazolyl,1H-imidazoimidazolyl, imidazooxazolyl or imidazo[5,1-b]thiazolyl. Insome embodiments, R is an optionally substituted 5,6-fused heteroarylring having four heteroatoms independently selected from nitrogen,oxygen, and sulfur. In some embodiments, R is an optionally substituted5,6-fused heteroaryl ring having five heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur.

In some embodiments, R is an optionally substituted 5,6-fused heteroarylring having 1-5 heteroatoms independently selected from nitrogen,oxygen, and sulfur. In other embodiments, R is an optionally substituted5,6-fused heteroaryl ring having 1-2 heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur. In certain embodiments, R is anoptionally substituted 5,6-fused heteroaryl ring having one heteroatomindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, R is optionally substituted indolyl. In some embodiments, Ris optionally substituted benzofuranyl. In some embodiments, R isoptionally substituted benzo[b]thienyl. In certain embodiments, R is anoptionally substituted 5,6-fused heteroaryl ring having two heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, R is optionally substituted azaindolyl. In someembodiments, R is optionally substituted benzimidazolyl. In someembodiments, R is optionally substituted benzothiazolyl. In someembodiments, R is optionally substituted benzoxazolyl. In someembodiments, R is an optionally substituted indazolyl. In certainembodiments, R is an optionally substituted 5,6-fused heteroaryl ringhaving three heteroatoms independently selected from nitrogen, oxygen,and sulfur. In some embodiments, R is optionally substitutedoxazolopyridiyl, thiazolopyridinyl or imidazopyridinyl. In certainembodiments, R is an optionally substituted 5,6-fused heteroaryl ringhaving four heteroatoms independently selected from nitrogen, oxygen,and sulfur. In some embodiments, R is optionally substituted purinyl,oxazolopyrimidinyl, thiazolopyrimidinyl, oxazolopyrazinyl,thiazolopyrazinyl, imidazopyrazinyl, oxazolopyridazinyl,thiazolopyridazinyl or imidazopyridazinyl. In certain embodiments, R isan optionally substituted 5,6-fused heteroaryl ring having fiveheteroatoms independently selected from nitrogen, oxygen, and sulfur.

In certain embodiments, R is an optionally substituted 6,6-fusedheteroaryl ring having 1-5 heteroatoms independently selected fromnitrogen, oxygen, and sulfur. In some embodiments, R is an optionallysubstituted 6,6-fused heteroaryl ring having 1-2 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In otherembodiments, R is an optionally substituted 6,6-fused heteroaryl ringhaving one heteroatom selected from nitrogen, oxygen, and sulfur. Insome embodiments, R is optionally substituted quinolinyl. In someembodiments, R is optionally substituted isoquinolinyl. In someembodiments, R is an optionally substituted 6,6-fused heteroaryl ringhaving two heteroatoms independently selected from nitrogen, oxygen, andsulfur. In some embodiments, R is optionally substituted quinazolinyl,phthalazinyl, quinoxalinyl or naphthyridinyl. In some embodiments, R isan optionally substituted 6,6-fused heteroaryl ring having threeheteroatoms independently selected from nitrogen, oxygen, and sulfur. Insome embodiments, R is optionally substituted pyridopyrimidinyl,pyridopyridazinyl, pyridopyrazinyl, or benzotriazinyl. In someembodiments, R is an optionally substituted 6,6-fused heteroaryl ringhaving four heteroatoms independently selected from nitrogen, oxygen,and sulfur. In some embodiments, R is optionally substitutedpyridotriazinyl, pteridinyl, pyrazinopyrazinyl, pyrazinopyridazinyl,pyridazinopyridazinyl, pyrimidopyridazinyl or pyrimidopyrimidinyl. Insome embodiments, R is an optionally substituted 6,6-fused heteroarylring having five heteroatoms independently selected from nitrogen,oxygen, and sulfur.

In some embodiments, R is optionally substituted C₆₋₃₀ arylaliphatic. Insome embodiments, R is optionally substituted C₆₋₂₀ arylaliphatic. Insome embodiments, R is optionally substituted C₆₋₁₀ arylaliphatic. Insome embodiments, an aryl moiety of the arylaliphatic has 6, 10, or 14aryl carbon atoms. In some embodiments, an aryl moiety of thearylaliphatic has 6 aryl carbon atoms. In some embodiments, an arylmoiety of the arylaliphatic has 10 aryl carbon atoms. In someembodiments, an aryl moiety of the arylaliphatic has 14 aryl carbonatoms. In some embodiments, an aryl moiety is optionally substitutedphenyl.

In some embodiments, R is optionally substituted C₆₋₃₀arylheteroaliphatic having 1-10 heteroatoms independently selected fromoxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, Ris optionally substituted C₆₋₃₀ arylheteroaliphatic having 1-10heteroatoms independently selected from oxygen, nitrogen, and sulfur. Insome embodiments, R is optionally substituted C₆₋₂₀ arylheteroaliphatichaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon. In some embodiments, R is optionallysubstituted C₆₋₂₀ arylheteroaliphatic having 1-10 heteroatomsindependently selected from oxygen, nitrogen, and sulfur. In someembodiments, R is optionally substituted C₆₋₁₀ arylheteroaliphatichaving 1-5 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon. In some embodiments, R is optionallysubstituted C₆₋₁₀ arylheteroaliphatic having 1-5 heteroatomsindependently selected from oxygen, nitrogen, and sulfur.

In some embodiments, two R groups are optionally and independently takentogether to form a covalent bond. In some embodiments, —C═O is formed.In some embodiments, —C═C— is formed. In some embodiments, —C≡C— isformed.

In some embodiments, two or more R groups on the same atom areoptionally and independently taken together with the atom to form anoptionally substituted, 3-30 membered, monocyclic, bicyclic orpolycyclic ring having, in addition to the atom, 0-10 heteroatomsindependently selected from oxygen, nitrogen, sulfur, phosphorus andsilicon. In some embodiments, two or more R groups on the same atom areoptionally and independently taken together with the atom to form anoptionally substituted, 3-20 membered monocyclic, bicyclic or polycyclicring having, in addition to the atom, 0-10 heteroatoms independentlyselected from oxygen, nitrogen, sulfur, phosphorus and silicon. In someembodiments, two or more R groups on the same atom are optionally andindependently taken together with the atom to form an optionallysubstituted, 3-10 membered monocyclic, bicyclic or polycyclic ringhaving, in addition to the atom, 0-5 heteroatoms independently selectedfrom oxygen, nitrogen, sulfur, phosphorus and silicon. In someembodiments, two or more R groups on the same atom are optionally andindependently taken together with the atom to form an optionallysubstituted, 3-6 membered monocyclic, bicyclic or polycyclic ringhaving, in addition to the atom, 0-3 heteroatoms independently selectedfrom oxygen, nitrogen, sulfur, phosphorus and silicon. In someembodiments, two or more R groups on the same atom are optionally andindependently taken together with the atom to form an optionallysubstituted, 3-5 membered monocyclic, bicyclic or polycyclic ringhaving, in addition to the atom, 0-3 heteroatoms independently selectedfrom oxygen, nitrogen, sulfur, phosphorus and silicon.

In some embodiments, two or more R groups on two or more atoms areoptionally and independently taken together with their intervening atomsto form an optionally substituted, 3-30 membered, monocyclic, bicyclicor polycyclic ring having, in addition to the intervening atoms, 0-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon. In some embodiments, two or more R groups on twoor more atoms are optionally and independently taken together with theirintervening atoms to form an optionally substituted, 3-20 memberedmonocyclic, bicyclic or polycyclic ring having, in addition to theintervening atoms, 0-10 heteroatoms independently selected from oxygen,nitrogen, sulfur, phosphorus and silicon. In some embodiments, two ormore R groups on two or more atoms are optionally and independentlytaken together with their intervening atoms to form an optionallysubstituted, 3-10 membered monocyclic, bicyclic or polycyclic ringhaving, in addition to the intervening atoms, 0-10 heteroatomsindependently selected from oxygen, nitrogen, sulfur, phosphorus andsilicon. In some embodiments, two or more R groups on two or more atomsare optionally and independently taken together with their interveningatoms to form an optionally substituted, 3-10 membered monocyclic,bicyclic or polycyclic ring having, in addition to the interveningatoms, 0-5 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon. In some embodiments, two or more Rgroups on two or more atoms are optionally and independently takentogether with their intervening atoms to form an optionally substituted,3-6 membered monocyclic, bicyclic or polycyclic ring having, in additionto the intervening atoms, 0-3 heteroatoms independently selected fromoxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments,two or more R groups on two or more atoms are optionally andindependently taken together with their intervening atoms to form anoptionally substituted, 3-5 membered monocyclic, bicyclic or polycyclicring having, in addition to the intervening atoms, 0-3 heteroatomsindependently selected from oxygen, nitrogen, sulfur, phosphorus andsilicon.

In some embodiments, heteroatoms in R groups, or in the structuresformed by two or more R groups taken together, are selected from oxygen,nitrogen, and sulfur. In some embodiments, a formed ring is 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20-membered. In someembodiments, a formed ring is saturated. In some embodiments, a formedring is partially saturated. In some embodiments, a formed ring isaromatic. In some embodiments, a formed ring comprises a saturated,partially saturated, or aromatic ring moiety. In some embodiments, aformed ring comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 aromatic ring atoms. In some embodiments, a formed contains nomore than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20aromatic ring atoms. In some embodiments, aromatic ring atoms areselected from carbon, nitrogen, oxygen and sulfur.

In some embodiments, a ring formed by two or more R groups (or two ormore groups selected from R and variables that can be R) taken togetheris a C₃₋₃₀ cycloaliphatic, C₆₋₃₀ aryl, 5-30 membered heteroaryl having1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon, or 3-30 membered heterocyclyl having 1-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon, ring as described for R, but bivalent ormultivalent.

In some embodiments, an amino acid of formula A-I is a compound havingthe structure of formula A-II:NH(R^(a1))-L^(a1)-C(-L^(a)-CH═CH₂)(R^(a3))-L^(a2)-COOH,   A-IIor a salt thereof, wherein each variable is independently as describedin the present disclosure.

In some embodiments, an amino acid of formula A-I is a compound havingthe structure of formula A-III:NH(R^(a1))—C(-L^(a)-CH═CH₂)(R^(a3))—COOH,   A-IIIor a salt thereof, wherein each variable is independently as describedin the present disclosure.

In some embodiments, L^(a) comprises at least one —N(R′)— wherein R′ isindependently as described in the present disclosure.

In some embodiments, an amino acid of formula A-I is a standard aminoacid. In some embodiments, an amino acid of formula A-I is selected fromTables A-I, A-II, and A-III:

TABLE A-I Exemplary amino acids (Fmoc-Protected).

TABLE A-II Exemplary amino acids (Fmoc-Protected).

TABLE A-III Exemplary amino acids (Fmoc-Protected).

In some embodiments, an amino acid is an alpha-amino acid. In someembodiments, an amino acid is an L-amino acid. In some embodiments, anamino acid is a D-amino acid. In some embodiments, the alpha-carbon ofan amino acid is achiral.

In some embodiments, an amino acid is a beta-amino acid. In someembodiments, an amino acid is beta-alanine.

In some embodiments, an amino acid is one whose residue is incorporatedin a peptide in Table 1.

In some embodiments, a provided amino acid sequence contains two or moreamino acid residues whose side chains are linked together to form one ormore staples. In some embodiments, a provided amino acid sequencecontains two or more amino acid residues, each of which independentlyhas a side chain comprising an olefin. In some embodiments, a providedamino acid sequence contains two or more amino acid residues, each ofwhich independently has a side chain comprising a terminal olefin. Insome embodiments, a provided amino acid sequence contains two and nomore than two amino acid residues, each of which independently has aside chain comprising an olefin. In some embodiments, a provided aminoacid sequence contains two and no more than two amino acid residues,each of which independently has a side chain comprising a terminalolefin. In some embodiments, a provided amino acid sequence comprises atleast one residue of an amino acid that comprises an olefin and anitrogen atom other than the nitrogen atom of its amino group. In someembodiments, a provided amino acid sequence comprises at least oneresidue of an amino acid that comprises a terminal olefin and a nitrogenatom other than the nitrogen atom of its amino group. In someembodiments, a provided amino acid sequence comprises at least oneresidue of an amino acid that has a side chain than comprises a terminalolefin and a nitrogen atom. In some embodiments, a provided amino acidsequence comprises at least one residue of an amino acid of formula A-I,wherein R^(a2) comprising an olefin and a —N(R′)— moiety, wherein R′ isas described in the present disclosure (including, in some embodiments,optionally taken together with R^(a3) and their intervening atoms toform an optionally substituted ring as described in the presentdisclosure). In some embodiments, R^(a2) comprising a terminal olefinand a —N(R′)— moiety wherein R′ is as described in the presentdisclosure. In some embodiments, a provided amino acid sequencecomprises at least one residue of an amino acid selected from Table A-I.In some embodiments, a provided amino acid sequence comprises at leastone residue of an amino acid selected from Table A-II. In someembodiments, a provided amino acid sequence comprises at least oneresidue of an amino acid selected from Table A-III. In some embodiments,two olefins from two side chains are linked together through olefinmetathesis to form a staple. In some embodiments, a staple is preferablyformed by side chains of amino acid residues that are not at thecorresponding positions of the Axin residues that interact withbeta-catenin. In some embodiments, a formed staple does not disruptinteraction between the peptide and beta-catenin.

In some embodiments, the present disclosure provides a peptidecomprising:[X¹]_(p1)[X²]_(p2)—X³X⁴X⁵X⁶X⁷X⁸X⁹X¹⁰—[X¹¹]_(p11)[X¹²]_(p12)[X¹³]_(p13),wherein:

each of p1, p2, p11, p12 and p13 is independently 0 or 1;

each of X, X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², and X¹³ isindependently an amino acid residue;

at least two of X, X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹²,and X¹³ comprise side chains that are optionally linked together to forma staple.

In some embodiments, a provided peptide is a stapled peptide, and atleast two of X¹ to X¹³ comprise side chains that are linked together toform a staple. In some embodiments, a provided peptide is an unstapledpeptide, wherein at least two of X¹ to X¹³ comprise side chains that canbe linked together to form a staple. In some embodiments, a stapledpeptide, or an unstapled peptide once stapled, interact withbeta-catenin at one or more beta-catenin sites that interact with Axin.In some embodiments, a stapled peptide, or an unstapled peptide oncestapled, interact with beta-catenin and compete with beta-catenininteraction with Axin or an Axin peptide.

In some embodiments, each of X¹ to X¹³ is independently an amino acidresidue of an amino acid having the structure of formula A-I.

In some embodiments, X^(i) and X^(i+m), each independently comprises aside chain that comprises an olefin, and the two side chains can belinked together to form a staple, e.g., a staple as described in thepresent disclosure, through olefin metathesis of the two olefins. Insome embodiments, both of the olefins are terminal olefins. In someembodiments, m is an integer of 3-12, and i is an integer of 1-18. Insome embodiments, m is an integer of 3-8, and i is an integer of 1-13.In some embodiments, at least one of X^(i) and X^(i+m) comprises a sidechain comprising an olefin and a nitrogen atom. In some embodiments, atleast one of X^(i) and X^(i+m) comprises —C(R^(2a))(R^(3a)) being—C(-L^(a)-R′)(R^(3a)), wherein at least one methylene unit of L^(a) isreplaced with —N(R′)— and R′ comprises an olefin. In some embodiments,at least one of X^(i) and X^(i+m) comprises —C(R^(2a))(R^(3a)) being—C(-L^(a)-CH═CH₂)(R^(3a)), wherein at least one methylene unit of L^(a)is replaced with —N(R′)—.

In some embodiments, i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, or 17. In some embodiments, i is 1. In some embodiments, i is 2.In some embodiments, i is 3. In some embodiments, i is 4. In someembodiments, i is 5. In some embodiments, i is 6. In some embodiments, iis 7. In some embodiments, i is 8. In some embodiments, i is 9. In someembodiments, i is 10. In some embodiments, i is 11. In some embodiments,i is 12. In some embodiments, i is 13. In some embodiments, i is 14. Insome embodiments, i is 15. In some embodiments, i is 16. In someembodiments, i is 17. In some embodiments, i is 18.

In some embodiments, m is 3. In some embodiments, m is 4. In someembodiments, m is 5. In some embodiments, m is 6. In some embodiments, mis 7. In some embodiments, m is 8. In some embodiments, m is 9. In someembodiments, m is 10. In some embodiments, m is 11. In some embodiments,m is 12.

In some embodiments, each of X^(i) and X^(i+m) is independently selectedfrom R₄, R₅, R₆, R₇, R₈, S₄, S₅, S₆, S₇, S₈, M_(A), M_(B), M_(C), M_(D),M_(E), M_(F), M_(G), M_(H), M_(I). In some embodiments, at least one ofX^(i) and X^(i+m) is independently selected from M_(A), M_(B), M_(C),M_(D), M_(E), M_(F), M_(G), M_(H), M_(I). In some embodiments, each ofX^(i) and X^(i+m) is independently selected from M_(A), M_(B), M_(C),M_(D), M_(E), M_(F), M_(G), M_(H), M_(I).

In some embodiments, X³ is a residue of an amino acid selected from R₄,R₅, R₆, R₇, R₈, S₄, S₅, S₆, S₇, S₈, M_(A), M_(B), M_(C), M_(D), M_(E),M_(F), M_(G), M_(H), and M_(I). In some embodiments, X³ is a residue ofan amino acid selected from R₄, R₅, R₆, R₇, R₈, S₄, S₅, S₆, S₇, and S₈.In some embodiments, wherein X³ is an amino acid residue of R₈. In someembodiments, wherein X³ is an amino acid residue of M_(G). In someembodiments, wherein X³ is an amino acid residue of R4. In someembodiments, X¹⁰ is a residue of an amino acid selected from R₄, R₅, R₆,R₇, R₈, S₄, S₅, S₆, S₇, S₈, M_(A), M_(B), M_(C), M_(D), M_(E), M_(F),M_(G), M_(H), and M_(I). In some embodiments, X¹⁰ is a residue of anamino acid selected from M_(A), M_(B), M_(C), M_(D), M_(E), M_(F),M_(G), M_(H), and M_(I). In some embodiments, X¹⁰ is a residue of R or ahomolog thereof. In some embodiments, X¹⁰ is a residue of R.

In some embodiments, X¹ is a residue of an amino acid selected from P,A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y, and α-methylproline. In some embodiments, X¹ is a residue of an amino acid selectedfrom P, A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y. Insome embodiments, X¹ is a residue of an amino acid selected from P, K,N, Q, R, Y, and α-methyl proline. In some embodiments, X¹ is a residueof an amino acid P. In some embodiments, X² is a residue of an aminoacid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W,and Y. In some embodiments, X² is a residue of an amino acid selectedfrom A, D, E, K, N, Q, and R. In some embodiments, X² is a residue of A.In some embodiments, X⁴ is a residue of an amino acid selected from I,F, H, L, V, homoleucine, tert-leucine, 3-cyclopropylalanine,3-cyclobutylalanine, 3-cyclopentylalanine, 3-cyclohexylalanine, andalpha-neopentylglycine. In some embodiments, X⁴ is a residue of an aminoacid selected from I, F, H, L, and V. In some embodiments, X⁴ is aresidue of an amino acid selected from I, L, V, homoleucine,tert-leucine, 3-cyclopropylalanine, 3-cyclobutylalanine,3-cyclopentylalanine, and alpha-neopentylglycine. In some embodiments,X⁴ is a residue of I. In some embodiments, X⁵ is a residue of an aminoacid selected from L, F, H, I, V, alpha-methyl leucine, homoleucine,tert-leucine, 3-cyclopropylalanine, 3-cyclobutylalanine,3-cyclopentylalanine, 3-cyclohexylalanine, and alpha-neopentylglycine.In some embodiments, X⁵ is a residue of an amino acid selected from L,F, H, I, and V. In some embodiments, X⁵ is a residue of an amino acidselected from L, I, V, alpha-methyl leucine, homoleucine, tert-leucine,3-cyclopropylalanine, 3-cyclobutylalanine, 3-cyclopentylalanine,3-cyclohexylalanine, and alpha-neopentylglycine. In some embodiments, X⁵is a residue of L. In some embodiments, X⁶ is a residue of an amino acidselected from D, A, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y,methionine sulfone, 2-aminoadipic acid, aspartic acid beta-methylester,aspartic acid beta-cyclohexylester, aspartic acid beta-benzylester,glutamic acid beta-methylester, glutamic acid beta-cyclohexylester, andglutamic acid beta-benzyl ester. In some embodiments, X⁶ is a residue ofan amino acid selected from D, A, E, F, H, I, K, L, M, N, P, Q, R, S, T,V, W, and Y. In some embodiments, X⁶ is a residue of an amino acidselected from D, E, H, N, Q, S, T, Y, methionine sulfone, 2-aminoadipicacid, aspartic acid beta-methylester, aspartic acidbeta-cyclohexylester, aspartic acid beta-benzylester, glutamic acidbeta-methylester, glutamic acid beta-cyclohexylester, and glutamic acidbeta-benzyl ester. In some embodiments, X⁶ is a residue of an amino acidselected from D, N, and T. In some embodiments, X⁷ is a residue of anamino acid selected from R₄, R₅, R₆, R₇, R₈, S₄, S₅, S₆, S₇, S₈, M_(A),M_(B), M_(C), M_(D), M_(E), M_(F), M_(G), M_(H), M_(I), A, D, E, F, H,I, K, L, M, N, P, Q, R, S, T, V, W, Y and alpha-methyl alanine. In someembodiments, X⁷ is a residue of an amino acid selected from A, D, E, F,H, I, K, L, M, N, P, Q, R, S, T, V, W, Y and alpha-methyl alanine. Insome embodiments, X⁷ is a residue of an amino acid selected from R₄, R₅,R₆, R₇, R₈, S₄, S₅, S₆, S₇, S₈, M_(A), M_(B), M_(C), M_(D), M_(E),M_(F), M_(G), M_(H), and M_(I). In some embodiments, X⁷ is a residue ofan amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T,V, W, and Y. In some embodiments, X⁷ is a residue of an amino acidselected from A, D, E, I, K, L, N, Q, R, S, T, V, W, Y and alpha-methylalanine. In some embodiments, X⁷ is a A or alpha-methyl alanine residue.In some embodiments, X⁸ is a residue of an amino acid selected from H,F, I, L, N, Q, V, 1-methylhistidine, 3-methylhistidine,3-(2-pyridyl)alanine, 3-(3-pyridyl)alanine, 3-(4-pyridyl)alanine,beta-2-furylalanine, beta-2-thienylalanine, 3-(2-tetrazolyl)alanine),and beta-4-thiazolylalanine. In some embodiments, X⁸ is a residue of anamino acid selected from H, F, I, L, N, Q, and V. In some embodiments,X⁸ is a residue of an amino acid selected from H, N, Q,1-methylhistidine, 3-methylhistidine, 3-(2-pyridyl)alanine,3-(3-pyridyl)alanine, 3-(4-pyridyl)alanine, beta-2-furylalanine,beta-2-thienylalanine, 3-(2-tetrazolyl)alanine), andbeta-4-thiazolylalanine. In some embodiments, X⁸ is a H residue. In someembodiments, X⁹ is a residue of an amino acid selected from I, V, F, H,L, homoleucine, tert-leucine, 3-cyclopropylalanine, 3-cyclobutylalanine,3-cyclopentylalanine, 3-cyclohexylalanine, and alpha-neopentylglycine.In some embodiments, X⁹ is a residue of an amino acid selected from I,V, F, H, and L. In some embodiments, X⁹ is a residue of an amino acidselected from I, V, L, homoleucine, tert-leucine, 3-cyclopropylalanine,3-cyclobutylalanine, 3-cyclopentylalanine, 3-cyclohexylalanine, andalpha-neopentylglycine. In some embodiments, X⁹ is a residue of an aminoacid selected from I and V. In some embodiments, X¹¹ is a residue of anamino acid selected from R, A, D, E, F, H, I, K, L, M, N, P, Q, S, T, V,W, Y, 3-(1-naphthylalanine), 2-aminoadipic acid, asymmetricdimethylarginine, symmetric dimethylarginine, homoarginine,N-epsilon-methyllysine, N-epsilon-dimethyllysine, andN-epsilon-trimethyllysine. In some embodiments, X¹¹ is a residue of anamino acid selected from R, A, D, E, F, H, I, K, L, M, N, P, Q, S, T, V,W, and Y. In some embodiments, X¹¹ is a residue of an amino acidselected from R, A, E, F, K, Q, S, V, Y, 3-(1-naphthylalanine),2-aminoadipic acid, asymmetric dimethylarginine, symmetricdimethylarginine, homoarginine, N-epsilon-methyllysine,N-epsilon-dimethyllysine, and N-epsilon-trimethyllysine. In someembodiments, X¹¹ is a residue of an amino acid selected from R, A, F, K,S, V, 3-(1-naphthylalanine), asymmetric dimethylarginine, symmetricdimethylarginine, homoarginine, and N-epsilon-methyllysine. In someembodiments, X¹² is a residue of an amino acid selected from V, F, H, I,L, alpha-methyl valine, alpha methyl leucine, homoleucine, tert-leucine,3-cyclopropylalanine, 3-cyclobutylalanine, 3-cyclopentylalanine,3-cyclohexylalanine, and alpha-neopentylglycine. In some embodiments,X¹² is a residue of an amino acid selected from V, F, H, I, and L. Insome embodiments, X¹² is a residue of an amino acid selected from I, A,L, V, alpha-methylleucine, homoleucine, tert-leucine,3-cyclopropylalanine, 3-cyclobutylalanine, 3-cyclopentylalanine,3-cyclohexylalanine, alpha-neopentylglycine, O-propargylserine,L-octylglycine, and L-alloisoleucine. In some embodiments, X¹² is aresidue of an amino acid selected from V, alpha-methyl valine, and alphamethyl leucine. In some embodiments, X¹³ is a residue of an amino acidselected from W, A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, Y,d-tryptophan, alpha-methyl tryptophan, 3-(1-naphthylalanine),3-(2-naphthylalanine), 4-chlorotryptophan, 5-chlorotryptophan,6-chlorotryptophan, 7-chlorotryptophan, 4-bromotryptophan,5-bromotryptophan, 6-bromotryptophan, 7-bromotryptophan,4-fluorotryptophan, 5-fluorotryptophan, 6-fluorotryptophan,7-fluorotryptophan, 1-methyltryptophan, 2-methyltryptophan,4-methyltryptophan, 5-methyltryptophan, 6-methyltryptophan,7-methyltryptophan, 2-hydroxytryptophan, 4-hydroxytryptophan,5-hydroxytryptophan, 6-hydroxytryptophan, 7-hydroxytryptophan,5-methoxytryptophan, 7-azatryptophan, 3-benzothienylalanine, and4-phenyl-L-phenylalanine. In some embodiments, X¹³ is a residue of anamino acid selected from W, A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T,V, and Y. In some embodiments, X¹³ is a residue of an amino acidselected from W, D, E, F, Y, d-tryptophan, alpha-methyl tryptophan,3-(1-naphthylalanine), 3-(2-naphthylalanine), 5-chlorotryptophan,6-chlorotryptophan, 7-chlorotryptophan, 5-bromotryptophan,6-bromotryptophan, 7-bromotryptophan, 5-fluorotryptophan,6-fluorotryptophan, 7-fluorotryptophan, 1-methyltryptophan,2-methyltryptophan, 5-methyltryptophan, 6-methyltryptophan,7-methyltryptophan, 2-hydroxytryptophan, 5-hydroxytryptophan,6-hydroxytryptophan, 7-hydroxytryptophan, 5-methoxytryptophan,7-azatryptophan, and 3-benzothienylalanine. In some embodiments, X¹³ isa residue of an amino acid selected from W, D-tryptophan, andalpha-methyl tryptophan.

In some embodiments, a provided peptide comprises SILDAHIQRVW (SEQ IDNO: 4) or a homolog thereof, therein at least two amino acid residues ofSILDAHIQRVW (SEQ ID NO: 4) or a homolog thereof is independentlyreplaced with X^(i) and X^(i+m). In some embodiments, a provided peptidecomprising X^(i)ILDAHIX^(i+m)RVW (SEQ ID NO: 5) or a homolog thereof. Insome embodiments, the side chains of X^(i) and X^(i+m) are linkedtogether through olefin metathesis to form a staple, e.g., one describedin the present disclosure. In some embodiments, one or more, or morethan half, or all of I, L, D, H, and V, corresponding to I472, L473,D474, H476, and V480 of Xenopus Axin are not replaced or replaced with ahomolog that has similar properties (e.g., a basic residue with a basichomolog, an acid residue with an acidic homolog, a hydrophobic residuewith a hydrophobic homolog, and/or an aromatic residue with an aromatichomolog). In some embodiments, one or more, or more than half, or all ofI, L, D, and H, corresponding to I472, L473, D474, and H476 of XenopusAxin are not replaced. In some embodiments, one of I, L, D, and H,corresponding to I472, L473, D474, and H476 of Xenopus Axin is notreplaced. In some embodiments, two of I, L, D, and H, corresponding toI472, L473, D474, and H476 of Xenopus Axin are not replaced. In someembodiments, three of I, L, D, and H, corresponding to I472, L473, D474,and H476 of Xenopus Axin are not replaced. In some embodiments, four ofI, L, D, and H, corresponding to I472, L473, D474, and H476 of XenopusAxin are not replaced. In some embodiments, all replacement, if any, areeach independently replaced with a homolog that has similar properties(e.g., a basic residue with a basic homolog, an acid residue with anacidic homolog, a hydrophobic residue with a hydrophobic homolog, and/oran aromatic residue with an aromatic homolog). In some embodiments, allreplacement, if any, are each independently replaced with a homolog,wherein if a basic residue is replaced, it is replaced with a basichomolog; if an acid residue, with an acidic homolog; if a hydrophobicresidue, with a hydrophobic homolog, and if an aromatic residue, with anaromatic homolog.

In some embodiments, a provide peptide has a sequence that is at least50%, 60%, 70%, 80%, 90%, or 95% homologous to PAR₈ILDAHVM_(B)RVW (SEQ IDNO: 6). In some embodiments, a provide peptide has a sequence that is atleast 50%, 60%, 70%, 80%, 90%, or 95% homologous to AR₈ILDAHIM_(B)RVW(SEQ ID NO: 7). In some embodiments, a provide peptide has a sequencethat is at least 50%, 60%, 70%, 80%, 90%, or 95% homologous toAM_(G)ILDAHIM_(B)RVW (SEQ ID NO: 8). In some embodiments, the homologyis at least 50%. In some embodiments, the homology is at least 60%. Insome embodiments, the homology is at least 70%. In some embodiments, thehomology is at least 80%. In some embodiments, the homology is at least80%. In some embodiments, the homology is at least 95%.

Exemplary peptides are extensively described in the present disclosure,e.g., in the Tables, Examples, etc. In some cases, a “-” may be includedin a compound (e.g., unstapled peptide, stapled peptide, etc.) ID numberafter “FP”. Unless otherwise specified, a number with a “-” after “FP”and a number without a “-” after “FP” refer to the same compound. Forexample, unless otherwise specified, FP-0996 (with a “-” after “FP”) andFP0996 (without a “-” after “FP”) refer to the same compound (in thiscase, the same peptide). In some embodiments, a provided peptide is apeptide of Table 1. In some embodiments, a provided stapled peptide is apeptide of Table 1. In some embodiments, a provided peptide is a peptidethat can undergo olefin metathesis to form a peptide of Table 1. In someembodiments, a provided stapled peptide is FPO217c. In some embodiments,a provided stapled peptide is FP0597c.

TABLE 1 Exemplary peptides. Part A: ID* SEQ ID NO: Sequence FP0001c 9Ac-P-Q-M_(C)-I-L-D-A-H-V-S₈-R-V-L-NH2 FP0003c 10Ac-P-A-M_(C)-I-L-D-A-H-V-S₈-R-V-L-NH2 FP0005c 11Ac-P-A-M_(C)-I-L-D-A-H-V-S₈-R-V-W-NH2 FP0006a 12Ac-P-A-M_(C)-I-L-D-A-H-V-S₈-R-V-W-NH2 FP0007c 13Ac-P-A-M_(C)-I-L-D-A-H-V-S₈-R-W-NH2 FP0009c 14Ac-P-A-M_(C)-I-A-D-A-H-V-S₈-R-V-W-NH2 FP0011c 15Ac-P-Q-R₈-I-L-D-A-H-V-M_(B)-R-V-L-NH2 FP0025c 16Ac-P-A-R₈-I-L-D-A-H-V-M_(B)-R-V-W-NH2 FP0098 17Ac-P-A-S-I-L-D-A-H-V-Q-R-V-W-NH2 FP0099 18Ac-P-A-M_(C)-I-L-D-A-H-V-S₈-R-V-W-NH2 FP0110 19Ac-P-E-S-I-L-D-E-H-V-Q-R-V-nL-K-NH2 FP0212s Isomer 2 20Ac-P-A-R₅-I-L-D-A-H-V-S₈-R-V-W-NH2 FP0216c 21Ac-P-A-R₈-I-L-T-A-H-I-M_(B)-R-V-W-NH2 FP0217a 22Ac-A-R₈-I-L-D-A-H-I-M_(B)-R-V-W-NH2 FP0217c 23Ac-A-R₈-I-L-D-A-H-I-M_(B)-R-V-W-NH2 c14-FP0217a 24Myr-A-R₈-I-L-D-A-H-I-M_(B)-R-V-W-NH2 c14-FP0217c 25Myr-A-R₈-I-L-D-A-H-I-M_(B)-R-V-W-NH2 c16-FP0217a 26Pal-A-R₈-I-L-D-A-H-I-M_(B)-R-V-W-NH2 FP0217c_bAfree 27bA-A-R₈-I-L-D-A-H-I-M_(B)-R-V-W-NH2 FP0217c_btn 28Btn-PEG3-A-R₈-I-L-D-A-H-I-M_(B)-R-V-W-NH2 FP0217c_c18a 29C18a-A-R₈-I-L-D-A-H-I-M_(B)-R-V-W-NH2 FP0217rc 30Ac-A-R₈-I-L-D-A-H-I-M_(B)-R-V-W-NH2 FP0217s Isomer 1 31Ac-A-R₈-I-L-D-A-H-I-S₅-R-V-W-NH2 FP0217s Isomer 2 32Ac-A-R₈-I-L-D-A-H-I-S₅-R-V-W-NH2 FP0217u 33Ac-A-R₈-I-L-D-A-H-I-M_(B)-R-V-W-NH2 FP0218c 34Ac-A-R₈-I-L-N-A-H-I-M_(B)-R-V-W-NH2 FP0219c 35Ac-A-R₈-I-L-T-A-H-I-M_(B)-R-V-W-NH2 FP0220c 36Ac-R₈-I-L-D-A-H-I-M_(B)-R-V-W-NH2 FP0221c 37Ac-R₈-I-L-N-A-H-I-M_(B)-R-V-W-NH2 FP0222c 38Ac-R₈-I-L-T-A-H-I-M_(B)-R-V-W-NH2 FP0223a 39Ac-P-A-M_(A)-I-L-D-A-H-V-S₈-R-V-W-NH2 FP0224a 40Ac-P-A-R₈-I-L-D-A-H-I-M_(A)-R-V-W-NH2 FP0243c 41Ac-A-M_(A)-I-L-pff-A-H-I-S₈-ADMA-V-W-NH2 FP0244c 42Ac-A-M_(A)-I-L-pff-A-H-I-S₈-Y-V-W-NH2 FP0247c 43Ac-A-M_(A)-I-L-ADMA-A-H-I-S₈-ADMA-V-W-NH2 FP0249c 44Ac-A-R₈-I-L-SDMA-A-H-I-M_(A)-ADMA-V-W-NH2 FP0250c 45Ac-A-M_(A)-I-L-ADMA-A-H-I-S₈-SDMA-V-W-NH2 FP0253c 46Ac-A-R₈-I-L-N-A-H-I-M_(A)-pff-V-W-NH2 FP0264c 47Ac-A-M_(A)-I-L-pff-A-H-I-S₈-A-V-W-NH2 FP0265c 48Ac-A-R₈-I-L-Y-A-H-I-M_(A)-Y-V-W-NH2 FP0268c 49Ac-A-M_(A)-I-L-N-A-H-I-S₈-ADMA-V-W-NH2 FP0269c 50Ac-A-R₈-I-L-ADMA-A-H-I-M_(A)-N-V-W-NH2 FP0270c 51Ac-A-M_(A)-I-L-ADMA-A-H-I-S₈-N-V-W-NH2 FP0271c 52Ac-A-M_(A)-I-L-N-A-H-I-S₈-SDMA-V-W-NH2 FP0272c 53Ac-A-R₈-I-L-ADMA-A-H-I-M_(A)-L-V-W-NH2 FP0273c 54Ac-A-M_(A)-I-L-SDMA-A-H-I-S₈-L-V-W-NH2 FP0274c 55Ac-A-M_(A)-I-L-ADMA-A-H-I-S₈-L-V-W-NH2 FP0278c 56Ac-A-M_(A)-I-L-Q-A-H-I-S₈-R-V-W-NH2 FP0279c 57Ac-A-R₈-I-L-N-A-H-I-M_(A)-Y-V-W-NH2 FP0280c 58Ac-A-M_(A)-I-L-Y-A-H-I-S₈-N-V-W-NH2 FP0281c 59Ac-A-R₈-I-L-Y-A-H-I-M_(A)-N-V-W-NH2 FP0282c 60Ac-A-M_(A)-I-L-N-A-H-I-S₈-Y-V-W-NH2 FP0284c 61Ac-A-M_(A)-I-L-SDMA-A-H-I-S₈-A-V-W-NH2 FP0285c 62Ac-A-M_(A)-I-L-D-A-H-I-S₈-R-V-W-NH2 FP0286c 63Ac-A-M_(A)-I-L-N-A-H-I-S₈-R-V-W-NH2 FP0290c 64Ac-A-M_(A)-I-L-N-A-H-I-S₈-cpa-V-W-NH2 FP0292c 65Ac-A-R₈-I-L-D-A-H-I-M_(A)-Q-V-W-NH2 FP0293c 66Ac-A-M_(A)-I-L-D-A-H-I-S₈-Q-V-W-NH2 FP0295c 67Ac-A-R₈-I-L-Q-A-H-I-M_(A)-N-V-W-NH2 FP0296c 68Ac-A-M_(A)-I-L-Q-A-H-I-S₈-N-V-W-NH2 FP0298c 69Ac-A-M_(A)-I-L-Q-A-H-I-S₈-T-V-W-NH2 FP0299c 70Ac-A-R₈-I-L-Q-A-H-I-M_(A)-T-V-W-NH2 FP0300c 71Ac-A-M_(A)-I-L-D-A-H-I-S₈-N-V-W-NH2 FP0302c 72Ac-A-M_(A)-I-L-N-A-H-I-S₈-L-V-W-NH2 FP0306c 73Ac-A-M_(A)-I-L-T-A-H-I-S₈-N-V-W-NH2 FP0317a 74Dodec-P-A-R₈-I-L-D-A-H-V-M_(B)-R-V-W-NH2 FP0318a 75Dec-P-A-R₈-I-L-D-A-H-V-M_(B)-R-V-W-NH2 FP0318c 76Dec-P-A-R₈-I-L-D-A-H-V-M_(B)-R-V-W-NH2 FP0321c 77Bua-P-A-R₈-I-L-D-A-H-V-M_(B)-R-V-W-NH2 FP0324c 780ct-P-A-R₈-I-L-D-A-H-V-M_(B)-R-V-W-1NH2 FP0325a 79Hex-P-A-R₈-I-L-D-A-H-V-M_(B)-R-V-W-NH2 FP0325c 80Hex-P-A-R₈-I-L-D-A-H-V-M_(B)-R-V-W-NH2 FP0327c 81Ac-P-A-R₈-I-A-D-A-H-V-M_(B)-R-V-W-NH2 FP0327c 81Ac-P-A-R₈-I-A-D-A-H-V-M_(B)-R-V-W-NH2 FP0335a 82Ac-A-M_(E)-I-L-D-A-H-I-S₈-R-V-W-NH2 FP0335c Isomer 1 83Ac-A-M_(E)-I-L-D-A-H-I-S₈-R-V-W-NH2 FP0335c Isomer 2 84Ac-A-M_(E)-I-L-D-A-H-I-S₈-R-V-W-NH2 FP0336c 85Ac-A-M_(E)-I-L-4FF-A-H-I-S₈-Y-V-W-NH2 FP0338c 86Ac-A-R₈-I-L-D-A-H-I-M_(D)-R-V-W-NH2 FP0344c 87Ac-A-R₈-I-L-4FF-A-H-I-M_(B)-R-V-W-NH2 FP0345c 88Ac-A-M_(A)-I-L-4FF-A-H-I-S₈-Y-V-W-NH2 FP0346c 89Ac-A-R₈-I-L-4FF-A-H-I-M_(A)-4FF-V-W-NH2 FP0349c 90Ac-A-R₈-I-L-MeY-A-H-I-M_(A)-4FF-V-W-NH2 FP0350c 91Ac-A-R₈-I-L-F-A-H-I-M_(B)-R-V-W-NH2 FP0352c 92Ac-A-R₈-I-L-F-A-H-I-M_(A)-4FF-V-W-NH2 FP0353c 93Ac-A-R₈-I-L-1NapA-A-H-I-M_(B)-R-V-W-NH2 FP0354c 94Ac-A-M_(A)-I-L-1NapA-A-H-I-S₈-Y-V-W-NH2 FP0355c 95Ac-A-R8-I-L-1NapA-A-H-I-M_(A)-4FF-V-W-NH2 FP0357c 96Ac-A-M_(A)-I-L-V-A-H-I-S₈-Y-V-W-NH2 FP0365c 97Ac-A-R₈-I-L-D-A-H-I-M_(B)-1NapA-V-W-NH2 FP0365c Isomer 1 98Ac-A-R₈-I-L-D-A-H-I-M_(B)-1NapA-V-W-NH2 FP0365c Isomer 2 99Ac-A-R₈-I-L-D-A-H-I-M_(B)-1NapA-V-W-NH2 FP0368c 100Ac-A-R₈-I-L-D-A-H-I-M_(B)-V-V-W-NH2 FP0369c 101Ac-A-M_(A)-I-L-4FF-A-H-I-S₈-V-V-W-NH2 FP0371c 102Ac-A-R₈-I-L-D-A-H-I-M_(B)-F-V-W-NH2 FP0380c 103Ac-A-R₈-I-L-D-A-H-I-M_(B)-R-Cha-W-NH2 FP0383c 104Ac-A-R₈-I-L-D-A-H-Cha-M_(B)-R-V-W-NH2 FP0391c 105Ac-A-R₈-I-L-2NapA-A-H-I-M_(A)-4FF-V-W-NH2 FP0395c 106Ac-A-R₈-I-L-Cha-A-H-I-M_(B)-R-V-W-NH2 FP0405c 107Ac-A-R₈-A-L-D-A-H-I-M_(B)-R-V-W-NH2 FP0406c 108Ac-A-R₈-I-A-D-A-H-I-M_(B)-R-V-W-NH2 FP0407c 109Ac-A-R₈-I-L-A-A-H-I-M_(B)-R-V-W-NH2 FP0408c 110Ac-A-R₈-I-L-D-A-A-I-M_(B)-R-V-W-NH2 FP0409a 111Ac-A-R₈-I-L-D-A-H-A-M_(B)-R-V-W-NH2 FP0409c 112Ac-A-R₈-I-L-D-A-H-A-M_(B)-R-V-W-NH2 FP0409c_free 113A-R₈-I-L-D-A-H-A-M_(B)-R-V-W-NH2 c16-FP0409a 114Pal-A-R₈-I-L-D-A-H-A-M_(B)-R-V-W-NH2 c16-FP0409c 115Pal-A-R₈-I-L-D-A-H-A-M_(B)-R-V-W-NH2 FP0410c 116Ac-A-R₈-I-L-D-A-H-I-M_(B)-A-V-W-NH2 FP0411c 117Ac-A-R₈-I-L-D-A-H-I-M_(B)-R-A-W-NH2 FP0412c 118Ac-A-R₈-I-L-D-A-H-I-M_(B)-R-V-A-NH2 FP0495a 119Ac-A-R₇-I-L-D-A-H-I-M_(D)-R-V-W-NH2 FP0495c 120Ac-A-R₇-I-L-D-A-H-I-M_(D)-R-V-W-NH2 FP0501c 121Ac-A-R₅-I-L-D-A-H-I-M_(F)-R-V-W-NH2 FP0502a 122Ac-A-R₆-I-L-D-A-H-I-M_(F)-R-V-W-NH2 FP0502c Isomer 1 123Ac-A-R₆-I-L-D-A-H-I-M_(F)-R-V-W-NH2 FP0502c Isomer 2 124Ac-A-R₆-I-L-D-A-H-I-M_(F)-R-V-W-NH2 FP0503a 125Ac-A-R₇-I-L-D-A-H-I-M_(F)-R-V-W-NH2 FP0503c 126Ac-A-R₇-I-L-D-A-H-I-M_(F)-R-V-W-NH2 FP0506a 127Ac-A-M_(I)-I-L-D-A-H-I-S₅-R-V-W-NH2 FP0506c Isomer 1 128Ac-A-M_(I)-I-L-D-A-H-I-S₅-R-V-W-NH2 FP0506c Isomer 2 129Ac-A-M_(I)-I-L-D-A-H-I-S₅-R-V-W-NH2 FP0507a 130Ac-A-M_(I)-I-L-D-A-H-I-S₆-R-V-W-NH2 FP0507c 131Ac-A-M_(I)-I-L-D-A-H-I-S₆-R-V-W-NH2 FP0509a 132Ac-A-R₄-I-L-D-A-H-I-M_(H)-R-V-W-NH2 FP0509c 133Ac-A-R₄-I-L-D-A-H-I-M_(H)-R-V-W-NH2 FP0510a 134Ac-A-R₅-I-L-D-A-H-I-M_(H)-R-V-W-NH2 FP0510c Isomer 1 135Ac-A-R₅-I-L-D-A-H-I-M_(H)-R-V-W-NH2 FP0510c Isomer 2 136Ac-A-R₅-I-L-D-A-H-I-M_(H)-R-V-W-NH2 FP0511a 137Ac-A-R₆-I-L-D-A-H-I-M_(H)-R-V-W-NH2 FP0511c Isomer 1 138Ac-A-R₆-I-L-D-A-H-I-M_(H)-R-V-W-NH2 FP0511c Isomer 2 139Ac-A-R₆-I-L-D-A-H-I-M_(H)-R-V-W-NH2 FP0516a Isomer 1 140Ac-A-R₇-I-L-D-A-H-I-M_(A)-R-V-W-NH2 FP0516a Isomer 2 141Ac-A-R₇-I-L-D-A-H-I-M_(A)-R-V-W-NH2 FP0516c 142Ac-A-R₇-I-L-D-A-H-I-M_(A)-R-V-W-NH2 FP0536c 143Ac-A-R₈-I-L-D-A-thi-I-M_(B)-R-V-W-NH2 FP0537c 144Ac-A-R₈-I-L-D-A-3pyr-I-M_(B)-R-V-W-NH2 FP0538c 145Ac-A-R₈-I-L-D-A-4pyr-I-M_(B)-R-V-W-NH2 FP0539c 146Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-R-V-W-NH2 FP0539c Isomer 1 147Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-R-V-W-NH2 FP0539c Isomer 2 148Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-R-V-W-NH2 FP0540c 149Ac-A-R₈-I-L-D-A-F-I-M_(B)-R-V-W-NH2 FP054 lc 150Ac-A-R₈-I-L-D-A-fur-I-M_(B)-R-V-W-NH2 FP0542c 151Ac-A-R₈-I-L-D-A-H-I-M_(B)-S-V-W-NH2 FP0554c Isomer 1 152Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-A-V-W-NH2 FP0554c Isomer 2 153Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-A-V-W-NH2 FP0555c Isomer 1 154Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-F-V-W-NH2 FP0555c Isomer 2 155Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-F-V-W-NH2 FP0556c Isomer 1 156Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-I-V-W-NH2 FP0556c Isomer 2 157Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-I-V-W-NH2 FP0557c Isomer 1 158Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-L-V-W-NH2 FP0557c Isomer 2 159Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-L-V-W-NH2 FP0558c Isomer 1 160Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-N-V-W-NH2 FP0558c Isomer 2 161Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-N-V-W-NH2 FP0559c Isomer 1 162Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-Q-V-W-NH2 FP0559c Isomer 2 163Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-Q-V-W-NH2 FP0560c Isomer 1 164Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-S-V-W-NH2 FP0560c Isomer 2 165Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-S-V-W-NH2 FP0561c Isomer 1 166Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-T-V-W-NH2 FP0561c Isomer 2 167Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-T-V-W-NH2 FP0562c Isomer 1 168Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-V-V-W-NH2 FP0562c Isomer 2 169Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-V-V-W-NH2 FP0563c Isomer 1 170Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-W-V-W-NH2 FP0563c Isomer 2 171Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-W-V-W-NH2 FP0564c Isomer 1 172Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-Y-V-W-NH2 FP0564c Isomer 2 173Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-Y-V-W-NH2 FP0565c Isomer 1 174Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-Cba-V-W-NH2 FP0565c Isomer 2 175Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-Cba-V-W-NH2 FP0566c Isomer 1 176Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-Cha-V-W-NH2 FP0567c Isomer 1 177Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-Nva-V-W-NH2 FP0567c Isomer 2 178Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-Nva-V-W-NH2 FP0568c Isomer 1 179Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-tLeu-V-W-NH2 FP0568c Isomer 2 180Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-tLeu-V-W-NH2 FP0569c Isomer 1 181Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-fur-V-W-NH2 FP0569c Isomer 2 182Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-fur-V-W-NH2 FP0570c Isomer 1 183Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-Aib-V-W-NH2 FP0570c Isomer 2 184Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-Aib-V-W-NH2 FP0571c Isomer 1 185Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-thi-V-W-NH2 FP0571c Isomer 2 186Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-thi-V-W-NH2 FP0572c Isomer 1 187Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-2pyr-V-W-NH2 FP0573c Isomer 1 188Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-3pyr-V-W-NH2 FP0573c Isomer 2 189Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-3pyr-V-W-NH2 FP0574c Isomer 1 190Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-cpa-V-W-NH2 FP0574c Isomer 2 191Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-cpa-V-W-NH2 FP0575c Isomer 1 192Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-MeY-V-W-NH2 FP0575c Isomer 2 193Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-MeY-V-W-NH2 FP0576c Isomer 1 194Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-4FF-V-W-NH2 FP0576c Isomer 2 195Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-4FF-V-W-NH2 FP0577c Isomer 1 196Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-1NapA-V-W-NH2 FP0578c Isomer 1 197Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-4MeF-V-W-NH2 FP0578c Isomer 2 198Ac-A-R₈-I-L-D-A-2pyr-I-M_(B)-4MeF-V-W-NH2 FP0587c 199Ac-A-M_(I)-I-L-D-A-H-I-M_(F)-R-V-W-NH2 FP0588c 200Ac-A-M_(I)-I-L-D-A-H-I-M_(G)-R-V-W-NH2 FP0594c 201Ac-A-M_(G)-I-L-D-A-H-I-M_(F)-R-V-W-NH2 FP0596c 202Ac-A-M_(G)-I-L-D-A-H-I-M_(E)-R-V-W-NH2 FP0597c 203Ac-A-M_(G)-I-L-D-A-H-I-M_(B)-R-V-W-NH2 FP0597c_c12 204Dodec-A-M_(G)-I-L-D-A-H-I-M_(B)-R-V-W-NH2 FP0597c_c8 205Oct-A-M_(G)-I-L-D-A-H-I-M_(B)-R-V-W-NH2 FP0598c 206Ac-A-M_(G)-I-L-D-A-H-I-M_(C)-R-V-W-NH2 FP0601c 207Ac-A-M_(E)-I-L-D-A-H-I-M_(F)-R-V-W-NH2 FP0604c 208Ac-A-M_(E)-I-L-D-A-H-I-M_(B)-R-V-W-NH2 FP0605c 209Ac-A-M_(E)-I-L-D-A-H-I-M_(C)-R-V-W-NH2 FP0611c 210Ac-A-M_(A)-I-L-D-A-H-I-M_(F)-R-V-W-NH2 FP0616c 211Ac-A-M_(A)-I-L-D-A-H-I-M_(C)-R-V-W-NH2 FP0617c 212Ac-A-M_(A)-I-L-D-A-H-I-M_(B)-R-V-W-NH2 FP0625c 213Ac-A-M_(A)-I-L-D-A-H-I-M_(I)-R-V-W-NH2 FP0626c 214Ac-A-M_(F)-I-L-D-A-H-I-M_(B)-R-V-W-NH2 FP0628_aib 215Ac-A-Aib-I-L-D-A-H-I-Aib-R-V-W-NH2 FP0629c 216Ac-A-R₈-I-L-M2O-A-H-I-M_(B)-R-V-W-NH2 FP0630c 217Ac-A-R₈-I-L-D-A-H-I-M_(B)-R-V-dW-NH2 FP0631c 218Ac-A-R₈-I-L-D-A-H-I-M_(B)-R-V-aMeW-NH2 FP0632c 219Ac-A-R₈-I-L-D-A-H-I-M_(B)-R-aMeV-W-NH2 FP0633c 220Ac-A-R₈-I-L-D-A-H-I-M_(B)-R-aMeL-W-NH2 FP0634c 221Ac-A-R₈-I-L-D-A-H-I-M_(B)-hArg-V-W-NH2 FP0635c 222Ac-A-R₈-I-L-D-A-H-I-M_(B)-K-V-W-NH2 FP0636c 223Ac-A-R₈-I-L-D-A-H-I-M_(B)-lmeK-V-W-NH2 FP0639c 224Ac-A-R₈-I-L-D-A-H-I-M_(B)-ADMA-V-W-NH2 FP0640c 225Ac-A-R₈-I-L-D-A-H-I-M_(B)-SDMA-V-W-NH2 FP0644c 226Ac-A-R₈-I-aMeL-D-A-H-I-M_(B)-R-V-W-NH2 FP0645c 227Ac-A-R₈-I-L-D-Aib-H-I-M_(B)-R-V-W-NH2 FP0721a 228Ac-P-Q-M_(A)-I-L-D-R₄-H-V-R-R-V-W-R-NH2 FP0721c 229Ac-P-Q-M_(A)-I-L-D-R₄-H-V-R-R-V-W-R-NH2 FP0723a 230Ac-P-Q-M_(A)-I-L-D-R₅-H-V-R-R-V-W-R-NH2 FP0723c 231Ac-P-Q-M_(A)-I-L-D-R₅-H-V-R-R-V-W-R-NH2 FP0724c 232Ac-P-Q-M_(A)-I-L-D-S₅-H-V-R-R-V-W-R-NH2 FP0725a 233Ac-P-Q-M_(A)-I-L-D-R₆-H-V-R-R-V-W-R-NH2 FP0725c 234Ac-P-Q-M_(A)-I-L-D-R₆-H-V-R-R-V-W-R-NH2 FP0727c 235Ac-P-Q-M_(A)-I-L-D-R₇-H-V-R-R-V-W-R-NH2 FP0728c 236Ac-P-Q-M_(A)-I-L-D-S₇-H-V-R-R-V-W-R-NH2 FP0731c 237Ac-P-Q-R₄-I-L-D-M_(A)-H-V-R-R-V-W-R-NH2 FP0733c 238Ac-P-Q-R₅-I-L-D-M_(A)-H-V-R-R-V-W-R-NH2 FP0734a 239Ac-P-Q-S₅-I-L-D-M_(A)-H-V-R-R-V-W-R-NH2 FP0734c 240Ac-P-Q-S₅-I-L-D-M_(A)-H-V-R-R-V-W-R-NH2 FP0735a 241Ac-P-Q-R₆-I-L-D-M_(A)-H-V-R-R-V-W-R-NH2 FP0735c 242Ac-P-Q-R₆-I-L-D-M_(A)-H-V-R-R-V-W-R-NH2 FP0736a 243Ac-P-Q-S₆-I-L-D-M_(A)-H-V-R-R-V-W-R-NH2 FP0736c 244Ac-P-Q-S₆-I-L-D-M_(A)-H-V-R-R-V-W-R-NH2 FP0738a 245Ac-P-Q-S₇-I-L-D-M_(A)-H-V-R-R-V-W-R-NH2 FP0738c 246Ac-P-Q-S₇-I-L-D-M_(A)-H-V-R-R-V-W-R-NH2 FP0743a 247Ac-P-Q-M_(C)-I-L-D-R₅-H-V-R-R-V-W-R-NH2 FP0743c 248Ac-P-Q-M_(C)-I-L-D-R₅-H-V-R-R-V-W-R-NH2 FP0745a 249Ac-P-Q-M_(C)-I-L-D-R₆-H-V-R-R-V-W-R-NH2 FP0745c 250Ac-P-Q-M_(C)-I-L-D-R₆-H-V-R-R-V-W-R-NH2 FP0751a 251Ac-P-Q-M_(B)-I-L-D-S₅-H-V-R-R-V-W-R-NH2 FP0751c 252Ac-P-Q-M_(B)-I-L-D-S₅-H-V-R-R-V-W-R-NH2 FP0752c 253Ac-P-Q-M_(B)-I-L-D-S₆-H-V-R-R-V-W-R-NH2 FP0753a 254Ac-P-Q-M_(B)-I-L-D-S₇-H-V-R-R-V-W-R-NH2 FP0758a 255Ac-P-Q-R₅-I-L-D-M_(B)-H-V-R-R-V-W-R-NH2 FP0758c 256Ac-P-Q-R₅-I-L-D-M_(B)-H-V-R-R-V-W-R-NH2 FP0761c 257Ac-P-Q-S₆-I-L-D-M_(B)-H-V-R-R-V-W-R-NH2 FP0763a 258Ac-P-Q-S₇-I-L-D-M_(B)-H-V-R-R-V-W-R-NH2 FP0763c 259Ac-P-Q-S₇-I-L-D-M_(B)-H-V-R-R-V-W-R-NH2 FP0765c 260Ac-P-Q-R₄-I-L-D-M_(C)-H-V-R-R-V-W-R-NH2 FP0766c 261Ac-P-Q-R₅-I-L-D-M_(C)-H-V-R-R-V-W-R-NH2 FP0767a 262Ac-P-Q-R₆-I-L-D-M_(C)-H-V-R-R-V-W-R-NH2 FP0767c 263Ac-P-Q-R₆-I-L-D-M_(C)-H-V-R-R-V-W-R-NH2 FP0768a 264Ac-P-Q-R₇-I-L-D-M_(C)-H-V-R-R-V-W-R-NH2 FP0768c 265Ac-P-Q-R₇-I-L-D-M_(C)-H-V-R-R-V-W-R-NH2 FP0776c 266Ac-P-Q-R₅-I-L-D-M_(G)-H-V-R-R-V-W-R-NH2 FP0776a 267Ac-P-Q-R₅-I-L-D-M_(G)-H-V-R-R-V-W-R-NH2 FP0777c 268Ac-P-Q-R₅-I-L-D-M_(I)-H-V-R-R-V-W-R-NH2 FP0777a 269Ac-P-Q-R₅-I-L-D-M_(I)-H-V-R-R-V-W-R-NH2 FP0778c 270Ac-P-Q-M_(D)-I-L-D-S₅-H-V-R-R-V-W-R-NH2 FP0779c 271Ac-P-Q-M_(F)-I-L-D-S₅-H-V-R-R-V-W-R-NH2 FP0780c 272Ac-P-Q-M_(H)-I-L-D-S₅-H-V-R-R-V-W-R-NH2 FP0782c 273Ac-P-Q-M_(G)-I-L-D-R₅-H-V-R-R-V-W-R-NH2 FP0783c 274Ac-P-Q-M_(I)-I-L-D-R₅-H-V-R-R-V-W-R-NH2 FP0783a 275Ac-P-Q-M_(I)-I-L-D-R₅-H-V-R-R-V-W-R-NH2 FP0787s 276Ac-P-Q-S₅-I-L-D-S₅-H-V-R-R-V-W-R-NH2 *u: unstapled; a, c and s: stapled,typically (i, i + 4) and (i, i + 7). Some stapled peptides may containtwo or more staples. For c, comprising a carbamate staple whichcomprises —N(R’)—C(O)—O—. For s, comprising a hydrocarbon staple whichcomprises neither —N(R')—C(O)—O— nor —N(R'). For a, compris- ing anamino staple which comprises —N(R')— which is not part of —N(R')—C(O)—O—(can be formed by removal of CO₂ from —N(R')—C(O)—O—). As appreciated bythose skilled in the art, a staple formed by two side chains eachindependently having the structure of —L^(a)—CH═CH₂ has the structure of—L^(a)—CH═CH—L^(a)—, wherein the two L^(a) are the same or different.For amino linker, —N(R')—C(O)—O— in L^(a) of the corresponding carbamatelinker is replaced with —N(R')—; r: olefin (—CH═CH—) in staple formed bymetathesis reduced to —CH₂—CH₂— (e.g., in rc). Non-natural amino acids(or protected form thereof) or modifications (or reagents forintroducing the modifications) in Table 1 (unless otherwise noted, allamino acids, if applicable, are L-amino acids): Myr = myristoyl Pal =palmitoyl Ac = acetyl nL = norleucine bA = beta-alanine Btn = biotinPEG3 = CAS# 557756-85-1 C18a = CAS# 871-70-5 pff =pentafluorophenylalanine ADMA = asymmetric dimethylarginine SDMA =symmetric dimethylarginine cpa = 3-cyclopropylalanine Dodec = dodecanoylDec = decanoyl Bua = butyryl Oct = octyl Hex = hexyl 4FF =4-fluorophenylalanine MeY = O-methyl tyrosine 1NapA =3-(1-naphthyl)-L-alanine 2NapA = 3-(2-naphthyl)-L-alanine Cha =3-cyclohexyl-L-alanine thi = beta-2-thienylalanine 2pyr =3-(2-pyridyl)-L-alanine 3pyr = 3-(3-pyridyl)-L-alanine 4pyr=3-(4-pyridyl)-L-alanine fur = 2-furyl-L-alanine cba =3-cyclobutylalanine Nva = norvaline tLeu = tert-leucine 4MeF =4-methyl-L-phenylalanine Aib = aminoisobutyric acid M2O = methioninesulfone dW = D-tryptophan aMeW = alpha-methyl-L-tryptophan aMeV =alpha-methyl-L-valine aMeL = alpha-methyl-L-leucine hArg = homoargininelmeK = N-epsilon-methyl-L-lysine FITC = fluorescein isothiocyanate NHBut= aminobutyric acid NHHex = aminohexanoic acid NHOct = aminooctanoicacid AzWT = azetidine-2-carboxylic acid Bip = 4-phenyl-L-phenylalanine5ClW = 5-chloro-L-tryptophan HOW= 5-hydroxy-L-tryptophan H2W=2,3-dihydro-L-tryptophan F3MeF = 4-trifluoromethyl-L-phenylalanine 4ClF= 4-chloro-L-phenylalanine

Part B - Amino acid sequence the same as FP0217. Amino Acid Amino Acid 21 (i + 7 ID Carbamate Staple Monomer A S₈ FP0512c

Monomer A S₇ FP0513c

Monomer A S₆ FP0514c

R₈ Monomer A FP0515c

R₇ Monomer A FP0516c

R₆ Monomer A FP0517c

Monomer E S₈ FP0335c

Monomer E S₇ FP0492c

Monomer E S₆ FP0491c

Monomer E S₅ FP0490c

R₈ Monomer D FP0338c

R₇ Monomer D FP0495c

R₆ Monomer D FP0494c

R₅ Monomer D FP0493c

Monomer G S₇ FP0499c

Monomer G S₆ FP0498c

Monomer G S₅ FP0497c

Monomer G S₄ FP0496c

R₇ Monomer F FP0503c

R₆ Monomer F FP0502c

R₅ Monomer F FP0501c

R₅ Monomer F FP0501c

R₄ Monomer F FP0500c

Monomer I S₆ FP0507c

Monomer I S₅ FP0506c

Monomer I S₄ FP0505c

Monomer I S₃ FP0504c

Monomer C S₈ FP0496c

Monomer C S₇ FP0485c

Monomer C S₆ FP0484c

Monomer C S₅ FP0483c

R₈ Monomer B FP0217c

R₇ Monomer B FP0489c

R₆ Monomer B FP0488c

R₅ Monomer B FP0487c

R₃ Monomer H FP0508c

R₄ Monomer H FP0509c

R₅ Monomer H FP0510c

R₆ Monomer H FP0511c

Monomer G S₇ FP0520c

R₇ Monomer F FP0521c

Monomer I S₆ FP0522c

R₆ Monomer H FP0523c

Monomer A Monomer B FP0617c

Monomer A Monomer C FP0616c

Monomer A Monomer A FP0615c

Monomer A Monomer F FP0611c

Monomer A Monomer E FP0623c

Monomer A Monomer G FP0624c

Monomer A Monomer I FP0625c

Monomer I Monomer A FP0529c

Monomer G Monomer A FP0599c

Monomer E Monomer A FP0606c

Monomer F Monomer A FP0627c

Monomer C Monomer A FP0618c

Monomer B Monomer A FP0619c

Monomer B Monomer B FP0613c

Monomer B Monomer F FP0609c

Monomer C Monomer F FP0610c

Monomer C Monomer C FP0612c

Monomer C Monomer B FP0614c

Monomer C Monomer E FP0620c

Monomer C Monomer G FP0621c

Monomer C Monomer I FP0622c

Monomer I Monomer F FP0587c

Monomer I Monomer G FP0588c

Monomer I Monomer E FP0589c

Monomer I Monomer B FP0590c

Monomer I Monomer C FP0591c

Monomer I Monomer I FP0593c

Monomer G Monomer F FP0594c

Monomer G Monomer G FP0595c

Monomer G Monomer E FP0596c

Monomer G Monomer B FP0597c

Monomer G Monomer C FP0598c

Monomer G Monomer I FP0600c

Monomer E Monomer F FP0601c

Monomer E Monomer G FP0602c

Monomer E Monomer E FP0603c

Monomer E Monomer B FP0604c

Monomer E Monomer C FP0605c

Monomer E Monomer I FP0607c

Monomer F Monomer F FP0608c

Monomer F Monomer B FP0626c

Amino Acid 1 Amino Acid 2 ID (i position) (i + 7 position) (Carbamate)ID Amino Staple R₇ Monomer A FP0516c FP0516a

Monomer E S₈ FP0335c FP0335a

R₈ Monomer D FP0338c FP0338a

R₇ Monomer D FP0495c FP0495a

R₇ Monomer F FP0503c FP0503a

R₆ Monomer F FP0502c FP0502a

Monomer I S₆ FP0507c FP0507a

Monomer I S₅ FP0506c FP0506a

R₈ Monomer B FP0217c FP0217a

R₄ Monomer H FP0509c FP0509a

R₅ Monomer H FP0510c FP0510a

R₆ Monomer H FP0511c FP0511a

b. Staples

In some embodiments, a staple is a linker that can link one amino acidresidue to another amino acid residue through bonding to peptidebackbone atoms of the amino acid residues and, as is understood by thoseskilled in the art, is not through the peptide backbone between thelinked amino acid residues. In some embodiments, a staple bonds to thepeptide backbone by replacing one or more hydrogen and/or substituents(e.g., side chains, O, etc.) on peptide backbone atoms (e.g., C, N,etc.).

In some embodiments, a staple may contribute to one or more propertiesand/or activities of a stapled peptide, reportedly through stabilizationof alpha-helix formed by a stapled peptide. Various types of stapleshave been reported and may be utilized in accordance with the presentdisclosure, for example, those described in U.S. Pat. No. 9,617,309, US2015-0225471, US 2016-0024153, US 2016-0215036, US2016-0244494,WO2017/062518, Azzarito et al, Nature Chemistry 5: 161-173 (2013), etc.,the staples of each of which are incorporated herein by reference.

In some embodiments, the present disclosure provides the insights thatstructural elements of staples (e.g., chemistry [e.g., hydrocarbon,non-hydrocarbon (e.g., comprising one or more heteroatoms orheteroatom-containing moieties such as amino, carbamate, etc.)],stereochemistry [e.g., stereochemistry of backbone atoms that staplesare connected to (e.g., if staples are connected to alpha-carbon atomsof amino acid residues, such carbon atoms being chiral (R S) orachiral)], positioning (to what amino acid residues/backbone atomsstaples are connected), sizes (length of staples), etc.) can havesignificant impact on properties and/or activities, and can be employedto design and/or optimize stapled peptides having significantly improvedproperties and/or activities (e.g., increased solubility, increased cellpermeability, increased stability, increased selectivity, loweredtoxicity, increased activity, etc.).

In some embodiments, a provided staple is a hydrocarbon staple. In someembodiments, a hydrocarbon staple comprises no chain heteroatoms whereina chain of a staple is the shortest covalent connection within thestaple from one end of the staple to the other end of the staple.

In some embodiments, a provided staple is a non-hydrocarbon staple. Insome embodiments, a non-hydrocarbon staple comprises one or more chainheteroatoms wherein a chain of a staple is the shortest covalentconnection within the staple from one end of the staple to the other endof the staple. In some embodiments, a non-hydrocarbon staple is acarbamate staple in that it comprises a —N(R′)—C(O)—O— moiety in itschain. In some embodiments, a non-hydrocarbon staple is an amino staplein that it comprises a —N(R′)— moiety in its chain, wherein the —N(R′)—moiety is not part of —N(R′)—C(O)—O—. In some embodiments, anon-hydrocarbon staple is an amino staple in that it comprises a —N(R′)—moiety in its chain, wherein the —N(R′)-moiety is not bonded to a carbonatom that additionally forms a double bond with a heteroatom (e.g.,—C(═O), —C(═S), —C(═N—R′), etc.).

In some embodiments, a provided stapled peptide comprises a staple whichstaple is L^(s), wherein L^(s) is -L^(s1)-L^(s2)-L^(s3)-, each ofL^(s1), L^(s2), and L^(s3) is independently L, wherein each L isindependently as described in the present disclosure. In someembodiments, a provided staple is L^(s).

In some embodiments, L^(s1) comprises at least one —N(R′)—, wherein R′is as described in the present disclosure. In some embodiments, the—N(R′)— is bonded to two carbon atoms, wherein neither of the two carbonatoms forms a double bond with a heteroatom. In some embodiments, the—N(R′)— is not bonded to —C(O)—. In some embodiments, the —N(R′)— is notbonded to —C(S)—. In some embodiments, the —N(R′)— is not bonded to—C(═NR′)—. In some embodiments, L^(s1) is -L′-N(R′)—, wherein L′ isoptionally substituted bivalent C₁-C₁₉ aliphatic. In some embodiments,L^(s1) is -L′-N(CH₃)—, wherein L′ is optionally substituted bivalentC₁-C₁₉ aliphatic.

In some embodiments, R′ is optionally substituted C₁₋₆ alkyl. In someembodiments, R′ is C₁₋₆ alkyl. In some embodiments, R′ is methyl. Insome embodiments, the peptide backbone atom to which L^(s1) is bonded isalso bonded to R¹, and R′ and R¹ are both R and are taken together withtheir intervene atoms to form an optionally substituted ring asdescribed in the present disclosure. In some embodiments, a formed ringhas no additional ring heteroatoms in addition to the nitrogen atom towhich R′ is bonded. In some embodiments, a formed ring is 3-membered. Insome embodiments, a formed ring is 4-membered. In some embodiments, aformed ring is 5-membered. In some embodiments, a formed ring is6-membered.

As defined herein, L′ is optionally substituted bivalent C₁-C₁₉aliphatic. In some embodiments, L′ is optionally substituted bivalentC₁-C₁₅ aliphatic. In some embodiments, L′ is optionally substitutedbivalent C₁-C₁₀ aliphatic. In some embodiments, L′ is optionallysubstituted bivalent C₁-C₉ aliphatic. In some embodiments, L′ isoptionally substituted bivalent C₁-C₈ aliphatic. In some embodiments, L′is optionally substituted bivalent C₁-C₇ aliphatic. In some embodiments,L′ is optionally substituted bivalent C₁-C₆ aliphatic. In someembodiments, L′ is optionally substituted bivalent C₁-C₅ aliphatic. Insome embodiments, L′ is optionally substituted bivalent C₁-C₄ aliphatic.In some embodiments, L′ is optionally substituted alkylene. In someembodiments, L′ is optionally substituted alkenylene. In someembodiments, L′ is unsubstituted alkylene. In some embodiments, L′ is—CH₂—. In some embodiments, L′ is —(CH₂)₂—. In some embodiments, L′ is—(CH₂)₃—. In some embodiments, L′ is —(CH₂)₄—. In some embodiments, L′is —(CH₂)₅—. In some embodiments, L′ is —(CH₂)₆—. In some embodiments,L′ is —(CH₂)₇—. In some embodiments, L′ is —(CH₂)s-. In someembodiments, L′ is bonded to a peptide backbone atom. In someembodiments, L′ is optionally substituted alkenylene. In someembodiments, L′ is unsubstituted alkenylene. In some embodiments, L′ is—CH₂—CH═CH—CH₂—.

In some embodiments, L^(s1) comprises at least one —N(R′)C(O)—, whereinR′ is as described in the present disclosure. In some embodiments,L^(s1) is -L′-N(R′)C(O)—, wherein each of L′ and R′ is independently asdescribed in the present disclosure. In some embodiments, L^(s1) is-L′-N(CH₃)C(O)—, wherein L′ is independently as described in the presentdisclosure.

In some embodiments, L^(s1) is a covalent bond.

In some embodiments, L^(s1) is L′, wherein L′ is as described in thepresent disclosure.

In some embodiments, L^(s2) is L, wherein L is as described in thepresent disclosure. In some embodiments, L^(s2) is L′, wherein L′ is asdescribed in the present disclosure. In some embodiments, L^(s2)comprises —CH₂—CH═CH—CH₂—. In some embodiments, L^(s2) is—CH₂—CH═CH—CH₂—. In some embodiments, L^(s2) comprises —(CH₂)₄—. In someembodiments, L^(s2) is —(CH₂)₄—.

In some embodiments, L^(s3) comprises at least one —N(R′)—, wherein R′is as described in the present disclosure. In some embodiments, the—N(R′)— is bonded to two carbon atoms, wherein neither of the two carbonatoms forms a double bond with a heteroatom. In some embodiments, the—N(R′)— is not bonded to —C(O)—. In some embodiments, the —N(R′)— is notbonded to —C(S)—. In some embodiments, the —N(R′)— is not bonded to—C(═NR′)—. In some embodiments, L^(s3) is -L′-N(R′)—, wherein L′ isoptionally substituted bivalent C₁-C₁₉ aliphatic. In some embodiments,L^(s3) is -L′-N(CH₃)—, wherein L′ is optionally substituted bivalentC₁-C₁₉ aliphatic.

In some embodiments, L^(s3) comprises at least one —N(R′)C(O)—, whereinR′ is as described in the present disclosure. In some embodiments,L^(s3) is -L′-N(R′)C(O)—, wherein each of L′ and R′ is independently asdescribed in the present disclosure. In some embodiments, L^(s3) is-L′-N(CH₃)C(O)—, wherein L′ is independently as described in the presentdisclosure.

In some embodiments, L^(s3) is L′, wherein L′ is as described in thepresent disclosure. In some embodiments, L^(s3) is optionallysubstituted alkylene. In some embodiments, L^(s3) is unsubstitutedalkylene.

In some embodiments, L^(s) comprises at least one —N(R′)—, wherein R′ isas described in the present disclosure. In some embodiments, the —N(R′)—is bonded to two carbon atoms, wherein neither of the two carbon atomsforms a double bond with a heteroatom. In some embodiments, the —N(R′)—is not bonded to —C(O)—. In some embodiments, the —N(R′)— is not bondedto —C(S)—. In some embodiments, the —N(R′)— is not bonded to —C(═NR′)—.In some embodiments, L^(s) comprises at least one —N(R′)C(O)—, whereinR′ is as described in the present disclosure.

In some embodiments, L comprises at least one —N(R′)—, wherein R′ is asdescribed in the present disclosure. In some embodiments, the —N(R′)— isbonded to two carbon atoms, wherein neither of the two carbon atomsforms a double bond with a heteroatom. In some embodiments, the —N(R′)—is not bonded to —C(O)—. In some embodiments, the —N(R′)— is not bondedto —C(S)—. In some embodiments, the —N(R′)— is not bonded to —C(═NR′)—.In some embodiments, L is -L′-N(R′)—, wherein L′ is optionallysubstituted bivalent C₁-C₁₉ aliphatic. In some embodiments, L is-L′-N(CH₃)—, wherein L′ is optionally substituted bivalent C₁-C₁₉aliphatic.

In some embodiments, L comprises at least one —N(R′)C(O)—, wherein R′ isas described in the present disclosure. In some embodiments, L is-L′-N(R′)C(O)—, wherein each of L′ and R′ is independently as describedin the present disclosure. In some embodiments, L is -L′-N(CH₃)C(O)—,wherein L′ is independently as described in the present disclosure.

In some embodiments, L is L′, wherein L′ is as described in the presentdisclosure. In some embodiments, L is optionally substituted alkylene.In some embodiments, L is unsubstituted alkylene.

In some embodiments, L is optionally substituted bivalent C₁-C₁₅aliphatic. In some embodiments, L is optionally substituted bivalentC₁-C₁₀ aliphatic. In some embodiments, L is optionally substitutedbivalent C₁-C₉ aliphatic. In some embodiments, L is optionallysubstituted bivalent C₁-C₈ aliphatic. In some embodiments, L isoptionally substituted bivalent C₁-C₇ aliphatic. In some embodiments, Lis optionally substituted bivalent C₁-C₆ aliphatic. In some embodiments,L is optionally substituted bivalent C₁-C₅ aliphatic. In someembodiments, L is optionally substituted bivalent C₁-C₄ aliphatic. Insome embodiments, L is optionally substituted alkylene. In someembodiments, L is optionally substituted alkenylene. In someembodiments, L is unsubstituted alkylene. In some embodiments, L is—CH₂—. In some embodiments, L is —(CH₂)₂—. In some embodiments, L is—(CH₂)₃—. In some embodiments, L is —(CH₂)₄—. In some embodiments, L is—(CH₂)₅—. In some embodiments, L is —(CH₂)₆—. In some embodiments, L is—(CH₂)₇—. In some embodiments, L is —(CH₂)s-. In some embodiments, L isbonded to a peptide backbone atom. In some embodiments, L is optionallysubstituted alkenylene. In some embodiments, L is unsubstitutedalkenylene. In some embodiments, L is —CH₂—CH═CH—CH₂—.

In some embodiments, one end of a staple is connected to an atom A^(n1)of the peptide backbone, wherein A^(n1) is optionally substituted withR¹ and is an atom of an amino acid residue at amino acid position n¹ ofthe peptide from the N-terminus, and the other end is connected to anatom A² of the peptide backbone, wherein A² is optionally substitutedwith R² (in some embodiments, R¹ and/or R² is R which can be hydrogen)and is an atom of an amino acid residue at amino acid position n² of thepeptide from the N-terminus, wherein each of n¹ and n² is independentlyan integer, and n²=n¹+m, wherein m is 3-12.

In some embodiments, m is 3. In some embodiments, m is 4. In someembodiments, m is 5. In some embodiments, m is 6. In some embodiments, mis 7. In some embodiments, m is 8. In some embodiments, m is 9. In someembodiments, m is 10. In some embodiments, m is 11. In some embodiments,a staple is referred to a (i, i+m) staple.

In some embodiments, A^(n1) is a carbon atom. In some embodiments,A^(n1) is achiral. In some embodiments, A^(n1) is chiral. In someembodiments, A^(n1) is R. In some embodiments, A^(n1) is S.

In some embodiments, A^(n2) is a carbon atom. In some embodiments,A^(n2) is achiral. In some embodiments, A^(n2) is chiral. In someembodiments, A^(n2) is R. In some embodiments, A^(n2) is S.

In some embodiments, A^(n1) is achiral and A² is achiral. In someembodiments, A^(n1) is achiral and A² is R. In some embodiments, A^(n1)is achiral and A² is S. In some embodiments, A^(n1) is R and A^(n2) isachiral. In some embodiments, A^(n1) is R and A^(n2) is R. In someembodiments, A^(n1) is R and A^(n2) is S. In some embodiments, A^(n1) isS and A² is achiral. In some embodiments, A^(n1) is S and A^(n2) is R.In some embodiments, A^(n1) is S and A² is S.

In some embodiments, provided stereochemistry at staple-backboneconnection points and/or combinations thereof, optionally together withone or more structural elements of provided peptide, e.g., staplechemistry (hydrocarbon, non-hydrocarbon), staple length, etc. canprovide various benefits, such as improved preparation yield, purity,and/or selectivity, improved properties (e.g., improved solubility,improved stability, lowered toxicity, improved selectivities, etc.),improved activities, etc. In some embodiments, provided stereochemistryand/or stereochemistry combinations are different from those typicallyused, e.g., those of U.S. Pat. No. 9,617,309, US 2015-0225471, US2016-0024153, US 2016-0215036, US2016-0244494, WO2017/062518, andprovided one or more of benefits described in the present disclosure.

In some embodiments, a staple can be of various lengths, in someembodiments, as represent by the number of chain atoms of a staple. Insome embodiments, a chain of a staple is the shortest covalentconnection in the staple from a first end (connection point with apeptide backbone) of a staple to a second end of the staple, wherein thefirst end and the second end are connected to two different peptidebackbone atoms. In some embodiments, a staple comprises 5-30 chainatoms, e.g., 5, 6, 7, 8, 9, or 10 to 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or 25 chain atoms. In some embodiments, a staplecomprises 5 chain atoms. In some embodiments, a staple comprises 6 chainatoms. In some embodiments, a staple comprises 7 chain atoms. In someembodiments, a staple comprises 8 chain atoms. In some embodiments, astaple comprises 9 chain atoms. In some embodiments, a staple comprises10 chain atoms. In some embodiments, a staple comprises 11 chain atoms.In some embodiments, a staple comprises 12 chain atoms. In someembodiments, a staple comprises 13 chain atoms. In some embodiments, astaple comprises 14 chain atoms. In some embodiments, a staple comprises15 chain atoms. In some embodiments, a staple comprises 16 chain atoms.In some embodiments, a staple comprises 17 chain atoms. In someembodiments, a staple comprises 18 chain atoms. In some embodiments, astaple comprises 19 chain atoms. In some embodiments, a staple comprises20 chain atoms. In some embodiments, a staple has a length of 5 chainatoms. In some embodiments, a staple has a length of 6 chain atoms. Insome embodiments, a staple has a length of 7 chain atoms. In someembodiments, a staple has a length of 8 chain atoms. In someembodiments, a staple has a length of 9 chain atoms. In someembodiments, a staple has a length of 10 chain atoms. In someembodiments, a staple has a length of 11 chain atoms. In someembodiments, a staple has a length of 12 chain atoms. In someembodiments, a staple has a length of 13 chain atoms. In someembodiments, a staple has a length of 14 chain atoms. In someembodiments, a staple has a length of 15 chain atoms. In someembodiments, a staple has a length of 16 chain atoms. In someembodiments, a staple has a length of 17 chain atoms. In someembodiments, a staple has a length of 18 chain atoms. In someembodiments, a staple has a length of 19 chain atoms. In someembodiments, a staple has a length of 20 chain atoms. In someembodiments, a staple has a length of 8-15 chain atoms. In someembodiments, a staple has 8-12 chain atoms. In some embodiments, astaple has 9-12 chain atoms. In some embodiments, a staple has 9-10chain atoms. In some embodiments, a staple has 8-10 chain atoms. In someembodiments, length of a staple can be adjusted according to thedistance of the amino acid residues it connects, for example, a longerstaple may be needed for a (i, i+7) staple than a (i, i+4) staple.Staple lengths may be otherwise described. For example, in someembodiments, staple lengths may be described as the total number ofchain atoms and non-chain ring atoms, where a non-chain ring atom is anatom of the staple which forms a ring with one or more chain atoms butis not a chain atom in that it is not within the shortest covalentconnection from a first end of the staple to a second end of the staple.In some embodiments, staples formed using Monomer A (which comprises aazetidine moiety), Monomer B (which comprises a pyrrolidine moiety),and/or Monomer C (which comprises a pyrrolidine moiety) may comprise oneor two non-chain ring atoms as illustrated in the exemplary stapledpeptides.

In some embodiments, a staple has no heteroatoms in its chain. In someembodiments, a staple comprises at least one heteroatom in its chain. Insome embodiments, a staple comprises at least one nitrogen atom in itschain.

In some embodiments, a staple is L^(s), wherein L^(s) is an optionallysubstituted, bivalent C₈₋₁₄ aliphatic group wherein one or moremethylene units of the aliphatic group are optionally and independentlyreplaced with —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—,—C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—,—S(O)₂N(R′)—, —C(O)S—, or —C(O)O—. In some embodiments, a staple isL^(s), wherein L^(s) is an optionally substituted, bivalent C₉₋₁₃aliphatic group wherein one or more methylene units of the aliphaticgroup are optionally and independently replaced with —C(R′)₂—, -Cy-,—O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—,or —C(O)O—. In some embodiments, a staple is L^(s), wherein L^(s) is anoptionally substituted, bivalent C₁₀₋₁₅ aliphatic group wherein one ormore methylene units of the aliphatic group are optionally andindependently replaced with —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, or —C(O)O—. In some embodiments,a staple is L^(s), wherein L^(s) is an optionally substituted, bivalentC₁₁₋₁₄ aliphatic group wherein one or more methylene units of thealiphatic group are optionally and independently replaced with —C(R′)₂—,-Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—,or —C(O)O—. In some embodiments, a staple is a (i, i+4) staple in thatnot including the two amino acid residues that are directly connected tothe staple, there are three amino acid residues between the two aminoacid residues that are directly connected to the staple. In someembodiments, a staple is a (i, i+7) staple in that not including the twoamino acid residues that are directly connected to the staple, there aresix amino acid residues between the two amino acid residues that aredirectly connected to the staple.

In some embodiments, for each of L^(s), L^(s1), L^(s2), and L^(s3), anyreplacement of methylene units, if any, is replaced with —N(R′)— or—N(R′)—C(O)—.

In some embodiments, an olefin in a staple is a Z-olefin. In someembodiments, an olefin in a staple in an E-olefin. In some embodiments,a provided composition comprises stapled peptides comprising a staplethat contains a Z-olefin and stapled peptides comprising a staple thatcontains an E-olefin. In some embodiments, a provided compositioncomprises stapled peptides comprising a staple that contains a Z-olefin.In some embodiments, a provided composition comprises stapled peptidescomprising a staple that contains an E-olefin. In some embodiments,otherwise identical stapled peptides that differ only in the E Zconfiguration of staple olefin demonstrate different properties and/oractivities as demonstrated herein. In some embodiments, stapled peptideswith E-olefin in a staple may provide certain desirable propertiesand/or activities given the context. In some embodiments, stapledpeptides with Z-olefin in a staple may provide certain desirableproperties and/or activities given the context.

In some embodiments, two staples may be bonded to the same atom of thepeptide backbone, forming a “stitch” structure.

In some embodiments, a staple is Pro-lock in that one end of the stapleis bonded to the alpha-carbon of a proline residue.

In some embodiments, an exemplary staple is a staple as illustratedbelow in Tables S-1, S-2, S-3, and S-4 (with exemplary peptide backboneillustrated for clarity (can be applied to other peptide backbone), Xbeing amino acid residues). In some embodiments, the olefin is Z. Insome embodiments, the olefin is E. In some embodiments, an (i, i+4)staple is selected from Table S-1. In some embodiments, an (i, i+4)staple is selected from Table S-2. In some embodiments, an (i, i+7)staple is selected from Table S-3. In some embodiments, an (i, i+7)staple is selected from Table S-4.

TABLE S-1 Exemplary staples.

TABLE S-2 Exemplary staples.

TABLE S-3 Exemplary staples.

TABLE S-4 Exemplary staples.

c. Modifications and Conjugations

In some embodiments, a provided peptide is optionally modified at itsbackbone, side chain, N-terminus and/or C-terminus, and is optionallyconjugated to a second entity. Various modifications and/or conjugationsare known in the art and can be utilized in accordance with the presentdisclosure.

In some embodiments, a provided peptide is capped. In some embodiments,a provided peptide is capped at the N-terminus. In some embodiments, aprovided peptide is capped by an amidation reaction which convert theN-terminal —NH₂ into an amide. In some embodiments, the capping isacetylation.

In some embodiments, a modification and/or conjugation is to incorporatea targeting moiety, e.g., those can facilitate delivery to certaincells, organs, and/or organisms.

In some embodiments, a second entity is a ligand, e.g., a ligand for aprotein receptor or an enzyme. In some embodiments, a ligand is acarbohydrate. In some embodiments, a modification is glycosylation. Insome embodiments, a second entity for conjugation is a carbohydrate. Insome embodiments, a carbohydrate is GalNac. In some embodiments, asecond entity is a protein ligand.

In some embodiments, a provided peptide is conjugated to a lipid moiety,e.g., through coupling with a fatty acid with an N-terminus. In someembodiments, a lipid moiety is or comprises an optionally substitutedC₅-C₁₀₀ aliphatic. In some embodiments, a lipid moiety is or comprisesan unsubstituted C₅-C₁₀₀ aliphatic. In some embodiments, a lipid moietyis decanoyl, dodecanoyl, myristoyl, octyl, or palmitoyl.

In some embodiments, a provided peptide is conjugated to a degradationsignal/entity. In some embodiments, a provided peptide is conjugated toa ligand for an E3 ubiquitin ligase.

In some embodiments, a provided peptide is conjugated to another peptideor protein. In some embodiments, a provided peptide is conjugated toanother stapled peptide that interacts with beta-catenin at a differentsite than the provide peptide. In some embodiments, a provided peptideis conjugated to another stapled peptide that interacts withbeta-catenin but does not compete with the provided peptide forbeta-catenin binding.

In some embodiments, a provided stapled peptide comprises a helix in its3-dimensional structure. In some embodiments, a provided stapled peptidecan form an alpha-helix.

d. Properties and Activities

As demonstrated in the present disclosure, provided technologies cansignificantly improve properties and/or activities of stapled peptides.

In some embodiments, a provided peptide can form a helix structure.

In some embodiments, a provided peptide binds to beta-catenin. In someembodiments, a provided peptide has a Kd of no greater than 0.001,0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 2, 3, 4, 5, or 10 uM for beta-catenin. In someembodiments, a provided peptide has a Kd of no greater than 0.01 uM. Insome embodiments, a provided peptide has a Kd of no greater than 0.05uM. In some embodiments, a provided peptide has a Kd of no greater than0.1 uM. In some embodiments, a provided peptide has a Kd of no greaterthan 0.2 uM. In some embodiments, a provided peptide has a Kd of nogreater than 0.5 uM. In some embodiments, a provided peptide has a Kd ofno greater than 1 uM. Various technologies can be utilized in accordancewith the present disclosure to assess Kd, for example, fluorescencepolarization, surface plasmon resonance, TR-FRET, etc.

In some embodiments, provided technologies provide improved stability.One challenge of using peptide as therapeutics is that peptides can bereadily degraded when administered to a subject. Among other things, thepresent disclosure provides stapled peptides with greatly improvedpharmacokinetics profiles. In some embodiments, provided stapledpeptides have significantly improved half-life.

In some embodiments, provided technologies greatly improved solubilityof stapled peptides. Among other things, the present disclosurerecognize that a significant challenge of using stapled peptides is thatstapled peptides, for example, those comprising hydrocarbon staples, mayhave low solubility in aqueous solutions, thereby complicatingformulation and delivery. In some embodiments, the present disclosureprovides stapled peptides with staples comprising —N(R′)— and/or—N(R′)—C(O)— moieties, which have improved solubility compared tostapled peptides that are otherwise identical but comprise hydrocarbonstaples instead of staples comprising —N(R′)— and/or —N(R′)—C(O)—moieties. In some embodiments, provided stapled peptides comprisingstaples that comprise —N(R′)— and/or —N(R′)—C(O)— moieties have asolubility of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,210, 220, 230, 240, or 250 uM in DPBS (per liter, 8 g sodium chloride,0.2 g potassium phosphate, monobasic, 1.15 g sodium phosphate, dibasic,and 0.2 g potassium chloride). In some embodiments, the solubility is atleast 1 uM in DPBS. In some embodiments, the solubility is at least 2 uMin DPBS. In some embodiments, the solubility is at least 3 uM in DPBS.In some embodiments, the solubility is at least 4 uM in DPBS. In someembodiments, the solubility is at least 5 uM in DPBS. In someembodiments, the solubility is at least 6 uM in DPBS. In someembodiments, the solubility is at least 7 uM in DPBS. In someembodiments, the solubility is at least 8 uM in DPBS. In someembodiments, the solubility is at least 9 uM in DPBS. In someembodiments, the solubility is at least 10 uM in DPBS. In someembodiments, the solubility is at least 20 uM in DPBS. In someembodiments, the solubility is at least 30 uM in DPBS. In someembodiments, the solubility is at least 40 uM in DPBS. In someembodiments, the solubility is at least 50 uM in DPBS. In someembodiments, the solubility is at least 60 uM in DPBS. In someembodiments, the solubility is at least 70 uM in DPBS. In someembodiments, the solubility is at least 80 uM in DPBS. In someembodiments, the solubility is at least 90 uM in DPBS. In someembodiments, the solubility is at least 100 uM in DPBS. In someembodiments, the solubility is at least 120 uM in DPBS. In someembodiments, the solubility is at least 150 uM in DPBS. In someembodiments, the solubility is at least 180 uM in DPBS. In someembodiments, the solubility is at least 200 uM in DPBS. In someembodiments, the solubility is at least 220 uM in DPBS. In someembodiments, the solubility is at least 250 uM in DPBS. In someembodiments, provided stapled peptides can achieve improved propertiesand/or activities using fewer acidic or basic amino acid residues,which, among other things, are often used to improve solubility (e.g.,FP0597c v. StAx-35R, removal of the C-terminal R). Various methods canbe utilized in accordance with the present disclosure to assesssolubility, including those described in the examples.

Among other things, the present disclosure provides methods forimproving solubility of stapled peptides. In some embodiments, thepresent disclosure encompasses the recognition and positioning of astaple can be utilized to modulate solubility. In some embodiments, thepresent disclosure provides methods for increasing or decreasingsolubility of a stapled peptide by adjusting positioning of a staple. Asdemonstrate herein, structural similarly or otherwise identical stapledpeptides can have greatly increased solubility (e.g., see FP0597c (98uM) v. 7 FPO217c (7 uM)).

In some embodiments, provided stapled peptides with provided structuralfeatures, e.g., non-hydrocarbon staples (e.g., those comprising one ormore staples that comprises one or more —N(R′)—C(O)— or —N(R′)—), staplepositioning, connection stereochemistry, etc., provides improvedproperties and/or activities, e.g., increased cell permeability,increased cellular activities, etc., compared to an appropriatereference peptide which in some embodiments, is an unstapled peptidehaving the same sequence, or in some embodiments, is a stapled peptidethat is otherwise identical but have a different type of staple, e.g., ahydrocarbon staple. For example, as reported in Grossmann et al. PNAS109 17942-17947, a hydrocarbon-stapled peptide, StAx-33,(Ac-PEG1-PQS₅ILDS₅HVRRVWR (SEQ ID NO: 277)), was not cell-permeable anddid not exhibit cell-based activity; to obtain a cell-permeable stapledpeptide with cell-based activity, 3 amino acids were added to theN-terminus of the peptide and one other Q→R mutation was made. However,these modifications can negatively impact other properties of thepeptide. For example, the resulting peptide StAx-35R(Ac-PEG1-RRWPRS₅ILDS₅HVRRVWR (SEQ ID NO: 278)) had a reduced affinitycompared to StAx-33. In some embodiments, provided stapled peptides canachieve improved properties and/or activities without using conjugationwith other entries, e.g., PEG as in StAx-33 and StAx-35R (e.g., FP0597cv. StAx-35R). In some embodiments, provided stapled peptides can achieveimproved properties and/or activities using a shorter amino acidsequence (e.g., FP0597c or FP0025c v. StAx-35R). In some embodiments,provided stapled peptides can achieve improved properties and/oractivities using fewer acidic or basic amino acid residues, which, amongother things, are often used to improve solubility (e.g., FP0597c orFP0025c v. StAx-35R). In a competition fluorescence polarization assay,FP0025c displaced a labeled probe from the axin site of beta-cateninwith an EC₅₀<100 nM and showed better than 50% inhibition of signal at10 μM in a beta-catenin luciferase reporter assay.

In some embodiments, provided stapled peptides provide selectivity invarious aspects. In some embodiments, provided stapled peptidesselectively interacts with beta-catenin sites that interact with Axinover those sites that interact with BCL9. In some embodiments, providedstapled peptides competes with FITC-PEG1-PQ-S5-ILD-S5-HVRRVWR (SEQ IDNO: 1), (with a hydrocarbon staple formed by two S5 via olefinmetathesis) for interaction with beta-catenin, but not or to asignificantly less extent withAc-LSQEQLEHRERSLQTLRDIQRML-(2-Nal)-βA₂-K(FAM)-NH₂ (SEQ ID NO: 279). Insome embodiments, provided stapled peptides competes withFITC-bA-PQ-S5-ILD-S5-HVRRVWR (SEQ ID NO: 3) (with a hydrocarbon stapleformed by two S5 via olefin metathesis) for interaction withbeta-catenin, but not or to a significantly less extent withAc-LSQEQLEHRERSLQTLRDIQRML-(2-Nal)-βA₂-K(FAM)-NH₂ (SEQ ID NO: 279). Insome embodiments, a reference stapled peptide that interacts withbeta-catenin at sites that that interact with Axin isFITC-PEG1-PQ-S5-ILD-S5-HVRRVWR (SEQ ID NO: 1) (hydrocarbon staple formedby two S5 via olefin metathesis). In some embodiments, a referencestapled peptide that interacts with beta-catenin at sites that interactwith Axin is FITC-bA-PQ-S5-ILD-S5-HVRRVWR (SEQ ID NO: 3) (hydrocarbonstaple formed by two S5 via olefin metathesis). In some embodiments, areference stapled peptide that interacts with beta-catenin at sites thatinteract with BCL9 is Ac-LSQEQLEHRERSLQTLRDIQRML-(2-Nal)-βA₂-K(FAM)-NH₂(SEQ ID NO: 279). In some embodiments, a significantly less extent isEC50, e.g., as measured by competition assays described in the presentdisclosure, that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 20, 30, 40, 50, 100, 200, 300, 400, or 500 fold higher. In someembodiments, a fold is 5 fold. In some embodiments, a fold is 10 fold.In some embodiments, a fold is 20 fold. In some embodiments, a fold is50 fold. In some embodiments, a fold is 100 fold. In some embodiments, afold is 500 fold.

In some embodiments, provided stapled peptides provide more specificmodulation of beta-catenin target gene expression compared to areference Wnt pathway modulator (e.g., IWR-1, ICG-001, etc.). In someembodiments, provided stapled peptides decrease expression levels of oneor more beta-catenin target genes in a type of cells that comprisesaberrant Wnt/beta-catenin signaling pathway, while a reference Wntpathway modulator does not do so or do so to a less extent. In someembodiments, provided stapled peptides do not decrease, or decrease tomuch less extent, expression levels of one or more beta-catenin targetgenes compared to a reference agent in a type of cells that compriseswild-type Wnt/beta-catenin signaling pathway.

In some embodiments, provided stapled peptides have low toxicity, e.g.,non-specific toxicity, compared to an appropriate reference peptide. Insome embodiments, a reference peptide is a stapled peptide thatinteracts with one or more beta-catenin sites that interact with Axinand comprises a hydrocarbon staple, e.g., WO2017062518. In someembodiments, a provided stapled peptide has less than 10%, 15%, 20%,25%, 30%, 40%, 50% non-specific cytotoxicity at a concentration of noless than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 uM as measured by a LDHrelease assay compared to an appreciate positive reference. In someembodiments, a provided stapled peptide comprises a staple comprising a—N(R′)— or —N(R′)—C(O)— moiety, and has lower non-specific cytotoxicitycompared to a peptide comprising a hydrocarbon staple but is otherwiseof the identical structure when assayed under a comparable condition.

In some embodiments, provided stapled peptides modulate one or morefunctions of beta-catenin. In some embodiments, provided stapledpeptides modulate one or more functions of beta-catenin associated withAxin binding. In some embodiments, provided stapled peptides modulateone or more functions of beta-catenin associated with interactions atone or more sites that interact with Axin. In some embodiments, providedstapled peptides modulate beta-catenin target gene expression. In someembodiments, provided stapled peptides inhibit cancer cell growth. Insome embodiments, provided stapled peptides provide increased activitiescompared to an appropriate reference agent. In some embodiments, areference agent is a stapled peptide. In some embodiments, a referenceagent is a stapled peptide that interacts with beta-catenin sites thatinteract with Axin.

4. Production of Stapled Peptides

Various technologies are known in the art can be utilized in accordancewith the present disclosure to prepare provided stapled peptides,including those described in the methods. In many embodiments, peptidesare prepared on solid phase on a synthesizer using, typically, Fmocchemistry. In some embodiments, staples are formed by olefin metathesis.In some embodiments, a product double bond of metathesis isreduced/hydrogenated. In some embodiments, CO₂ are extruded from acarbamate moiety of a staple. In some embodiments, provided stapledpeptides are further modified, and/or conjugated to other entities.Conditions and/or reagents of these reactions are widely know in the artand can be performed in accordance with the present disclosure toprovide stapled peptides.

Properties and/or activities of provided stapled peptides can be readilyassessed in accordance with the present disclosure, for example, throughuse of one or more methods described in the examples.

In some embodiments, the present disclosure encompasses the recognitionthat structural elements of staples, e.g., size, chemistry,stereochemistry, etc., can significantly impact yields and/or purity ofstapling through olefin metathesis. As illustrated by exemplary dataprovided in the present disclosure, staples having certain structuralelements, e.g., size, chemistry, stereochemistry, etc., and/orcombination thereof, can facilitate production of provided stapledpeptides including higher yields, purity, and selectivity, etc. In someembodiments, the present disclosure provides beneficial structuralelements, e.g., size, chemistry, stereochemistry, etc., and/orcombination thereof, for example, those exemplified in the examples.

In some embodiments, the present disclosure provides the recognitionthat catalysts other than Grubbs I may provide better results, e.g.,yield, purity, selectivity, etc. for olefin metathesis. In someembodiments, the present disclosure demonstrates that Hoveyda-Grubbs IIcatalyst may provide better results, e.g., yield, purity, selectivity,etc. for olefin metathesis. In some embodiments, the present disclosureprovides methods for preparing a provided stapled peptide, comprisingproviding a Hoveyda-Grubbs II in an olefin metathesis reaction.

In some embodiments, technologies for preparing and/or assessingprovided stapled peptides include those described in U.S. Pat. No.9,617,309, US 2015-0225471, US 2016-0024153, US 2016-0215036,US2016-0244494, WO2017/062518, etc.

In some embodiments, a provided agent, e.g, a provided peptide, has apurity of 60%-100%. In some embodiments, a provided agent has a purityof at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99%. In some embodiments, a purity is at least 60%. Insome embodiments, a purity is at least 70%. In some embodiments, apurity is at least 80%. In some embodiments, a purity is at least 85%.In some embodiments, a purity is at least 90%. In some embodiments, apurity is at least 91%. In some embodiments, a purity is at least 92%.In some embodiments, a purity is at least 93%. In some embodiments, apurity is at least 94%. In some embodiments, a purity is at least 95%.In some embodiments, a purity is at least 96%. In some embodiments, apurity is at least 97%. In some embodiments, a purity is at least 98%.In some embodiments, a purity is at least 99%. In some embodiments, apurity is at least 99.5%.

In some embodiments, provided methods provide high yields. In someembodiments, a yield is 50%-100%. In some embodiments, a yield is atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99%. In some embodiments, a yield is at least 60%. In someembodiments, a yield is at least 65%. In some embodiments, a yield is atleast 70%. In some embodiments, a yield is at least 75%. In someembodiments, a yield is at least 80%. In some embodiments, a yield is atleast 85%. In some embodiments, a yield is at least 90%. In someembodiments, a yield is at least 91%. In some embodiments, a yield is atleast 92%. In some embodiments, a yield is at least 93%. In someembodiments, a yield is at least 94%. In some embodiments, a yield is atleast 95%. In some embodiments, a yield is at least 96%. In someembodiments, a yield is at least 97%. In some embodiments, a yield is atleast 98%. In some embodiments, a yield is at least 99%.

In some embodiments, a provided method delivers high E/Z selectivity forolefin. In some embodiments, provided selectivity favors the E isomer.In some embodiments, provided selectivity favors the Z isomer. In someembodiments, a E:Z ratio is at least 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, or 100:1. In someembodiments, a Z:E ratio is at least 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1,6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 80:1, 90:1, 95:1,99:1, or 100:1. In some embodiments, a ratio is at least 1:1. In someembodiments, a ratio is at least 1.5:1. In some embodiments, a ratio isat least 2:1. In some embodiments, a ratio is at least 3:1. In someembodiments, a ratio is at least 4:1. In some embodiments, a ratio is atleast 5:1. In some embodiments, a ratio is at least 6:1. In someembodiments, a ratio is at least 7:1. In some embodiments, a ratio is atleast 8:1. In some embodiments, a ratio is at least 9:1. In someembodiments, a ratio is at least 10:1. In some embodiments, a ratio isat least 20:1. In some embodiments, a ratio is at least 30:1. In someembodiments, a ratio is at least 40:1. In some embodiments, a ratio isat least 50:1. In some embodiments, a ratio is at least 80:1. In someembodiments, a ratio is at least 90:1. In some embodiments, a ratio isat least 95:1. In some embodiments, a ratio is at least 99:1. In someembodiments, a ratio is at least 100:1.

In some embodiments, a provide method comprises a period of time at atemperature higher than room temperature. In some embodiments, atemperature is about 25-200° C. In some embodiments, a temperature isabout 25° C. In some embodiments, a temperature is about 30° C. In someembodiments, a temperature is about 35° C. In some embodiments, atemperature is about 40° C. In some embodiments, a temperature is about45° C. In some embodiments, a temperature is about 50° C. In someembodiments, a temperature is about 55° C. In some embodiments, atemperature is about 60° C. In some embodiments, a temperature is about65° C. In some embodiments, a temperature is about 70° C. In someembodiments, a temperature is about 75° C. In some embodiments, atemperature is about 80° C. In some embodiments, a temperature is about85° C. In some embodiments, a temperature is about 90° C. In someembodiments, a temperature is about 95° C. In some embodiments, atemperature is about 100° C. In some embodiments, a temperature is about150° C. In some embodiments, a temperature is higher than about 150° C.

5. Uses

Among other things, provided stapled peptides interacts withbeta-catenin. In some embodiments, a condition, disorder, or disease isassociated with one or more components involved in Wnt/beta-cateninsignaling. In some embodiments, a condition, disorder, or disease isassociated with one or more beta-catenin functions. In some embodiments,a condition disorder or disease is associated with interactions betweenbeta-catenin and one or more beta-catenin sites that interact with oneor more proteins in Wnt/beta-catenin signaling. In some embodiments,provided stapled peptides compete with and/or otherwise interfere withor reduce binding between beta-catenin and Axin. In some embodiments, acondition disorder or disease is associated with interactions betweenbeta-catenin and one or more beta-catenin sites that interact with Axin.In some embodiments, a condition, disorder, or disease is associatedwith interactions with and one or more proteins that compete with Axinfor interaction with beta-catenin. In some embodiments, a providedstapled peptide antagonizes beta-catenin interaction with anotherprotein, such as TCF, whose one or more binding sites overlap with, orare in close proximity to, one or more beta-catenin sites that interactwith Axin or a provided stapled peptide. In some embodiments, acondition, disorder, or disease is associated with interactions betweenbeta-catenin and Axin. In some embodiments, provided stapled peptidesinteracts with beta-catenin at one or more beta-catenin sites thatinteracts with Axin. In some embodiments, provided stapled peptidesinhibit one or more Axin activities. In some embodiments, providedstapled peptides inhibit one or more Wnt/beta-catenin pathwayactivities.

In some embodiments, provided stapled peptides is useful for preventingand/or treating one or more beta-catenin-associated conditions,disorders, and/or diseases. In some embodiments, the present disclosureprovides a method for preventing or treating a beta-catenin-associatedcondition, disorder or disease, comprising administering to a subjectsusceptible to or suffering from provided stapled peptide or apharmaceutical composition thereof.

In some embodiments, a condition, disorder, or disease is selected fromcancer, cardiac disease, dilated cardiomyopathy, fetal alcohol syndrome,depression, and diabetes.

In some embodiments, a condition, disorder, or disease is a heartcondition, disorder, or disease.

In some embodiments, a condition, disorder, or disease is cancer. Insome embodiments a cancer is selected from: colon cancer, colorectalcancer, rectal cancer, prostate cancer familial adenomatous polyposis(FAP), Wilms Tumor, melanoma, hepatocellular carcinoma, ovarian cancer,endometrial cancer, medulloblastoma pilomatricomas, primaryhetpatocellular carcinoma, ovarial carcinoma, breast cancer, lungcancer, glioblastoma, pliomatrixoma, medulloblastoma, thyroid tumors,ovarian neoplasms. In some embodiments, a cancer is colorectal cancer.In some embodiments, a cancer is hepatocellular cancer. In someembodiments, a cancer is prostate cancer. In some embodiments, a canceris melanoma.

In some embodiments, a provided stapled peptide is administered incombination with an additional agent. In some embodiments, a providedstapled peptide is administered prior to, concurrently with, orsubsequent to an additional agent. In some embodiments, a providedstapled peptide is administered at the same time as an additional agent.In some embodiments, an additional agent is a therapeutic agent. In someembodiments, an additional agent may optionally be formulated with aprovided stapled peptide in the same pharmaceutical composition.

In some embodiments, an additional agent is a checkpoint inhibitor, anEGFR inhibitor, a VEGF inhibitor, a VEGFR inhibitor, a kinase inhibitor,or an anti-cancer drug.

In some embodiments, an additional agent is a checkpoint inhibitor. Insome embodiments, an additional agent is an immune oncology agent. Insome embodiments, an additional agent is an antibody against acheckpoint molecules. In some embodiments, an additional agent is anantibody of PD1, PDL-1, CTLA4, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3,TIM-s, C10orf54, etc. In some embodiments, an antibody is an anti-PD1antibody. In some embodiments, an antibody is an anti-PD-L1 antibody. Insome embodiments, an antibody is an anti-CTLA4.

In some embodiments, an additional agent is an EGFR inhibitor, e.g.,erlotinib, gefitinib, lapatinib, panitumumab, vandetanib, cetuximab,etc.

In some embodiments, an additional agent is an VEGF and/or VEGFRinhibitor, e.g., pazopanib, bevacizumab, sorafenib, sunitinib, axitinib,ponatinib, regorafenib, vandetanib, cabozantinib, ramucirumab,lenvatinib, ziv-aflibercept, etc.

In some embodiments, an additional agent is a kinase inhibitor. In someembodiments, an additional therapeutic agent is a chemotherapeuticagent. In some embodiments, an additional therapeutic agent is ananti-cancer drug, e.g., cyclophosphamide, methotrexate, 5-fluorouracil(5-FU), doxorubicin, mustine, vincristine, procarbazine, prednisolone,dacarbazine, bleomycin, etoposide, cisplatin, epirubicin, capecitabine,folinic acid, actinomycin, all-trans retinoic acid, azacitidine,azathioprine, bortezomib, carboplatin, chlorambucil, cytarabine,daunorubicin, docetaxel, doxifluridine, fluorouracil, gemcitabine,hydroxyurea, idarubicin, imatinib, irinotecan, mechlorethamine,mercaptopurine, mitoxantrone, paclitaxel, pemetrexed, teniposide,tioguanine, topotecan, valrubicin, vinblastine, vindesine, vinorelbine,oxaliplatin, etc.

In some embodiments, an additional agent is a stapled peptide. In someembodiments, an additional agent is a stapled peptide that interactswith beta-catenin that does not compete with binding betweenbeta-catenin and Axin. In some embodiments, an additional agent is astapled peptide that interacts with beta-catenin at one or more sitesthat interacts with BCL9.

In some embodiments, a provided stapled peptide is administered incombination with an additional therapy. In some embodiments, anadditional therapy is radiation therapy. In some embodiments, anadditional therapy is surgery.

6. Example Embodiments

Among other things, the present disclosure provides the followingExample Embodiments:

1. A peptide comprising:[X¹]_(p1)[X²]_(p2)—X³X⁴X⁵X⁶X⁷X⁸X⁹X¹⁰—[X¹¹]_(p11)[X¹²]_(p12)[X¹³]_(p13),wherein:

each of p1, p2, p11, p12 and p13 is independently 0 or 1;

each of X, X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², and X¹³ isindependently an amino acid residue;

at least two of X, X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹²,and X¹³ comprise side chains that are optionally linked together to forma staple.

2. The peptide of embodiment 1, wherein at least two of X¹ to X¹³ eachindependently comprise a side chain that comprise an olefin, wherein thetwo olefins can be connected together by olefin metathesis to form astaple.

3. The peptide of any one of the preceding embodiments, wherein sidechains of the at least two of X¹ to X¹³ are connected to form a staple.

4. The peptide of any one of the preceding embodiments, wherein each ofX¹ to X¹³ is independently a residue of an amino acid having thestructure of formula A-I.

5. The peptide of any one of the preceding embodiments, wherein each ofX¹ to X¹³ is independently a residue of an amino acid having thestructure of formula A-I and is an alpha amino acid.

6. The peptide of any one of the preceding embodiments, wherein each ofp1, p2, p11, p12 and p13 is independently 0.

7. The peptide of any one of embodiments 1-5, wherein each of p1, p2,p11, p12 and p13 is independently 1.

8. The peptide of any one of embodiments 1-5, wherein each of p1 is 0.

9. The peptide of any one of the preceding embodiments, wherein X³ is aresidue of an amino acid selected from R₄, R₅, R₆, R₇, R₈, S₄, S₅, S₆,S₇, S₈, M_(A), M_(B), M_(C), M_(D), M_(E), M_(F), M_(G), M_(H), andM_(I).

10. The peptide of any one of the preceding embodiments, wherein X³ is aresidue of an amino acid selected from R₄, R₅, R₆, R₇, R₈, S₄, S₅, S₆,S₇, and S₈.

11. The peptide of any one of the preceding embodiments, wherein X³ isan amino acid residue of R₈.

12. The peptide of any one of embodiments 1-9, wherein X³ is an aminoacid residue of M_(G).

13. The peptide of any one of embodiments 1-9, wherein X³ is an aminoacid residue of R₄.

14. The peptide of any one of the preceding embodiments, X¹⁰ is aresidue of an amino acid selected from R₄, R₅, R₆, R₇, R₈, S₄, S₅, S₆,S₇, S₈, M_(A), M_(B), M_(C), M_(D), M_(E), M_(F), M_(G), M_(H), andM_(I).

15. The peptide of any one of the preceding embodiments, X¹⁰ is aresidue of an amino acid selected from M_(A), M_(B), M_(C), M_(D),M_(E), M_(F), M_(G), M_(H), and M_(I).

16. The peptide of any one of embodiments 1-13, wherein X¹⁰ is a residueof R or a homolog thereof.

17. The peptide of any one of embodiments 1-13, wherein X¹⁰ is a residueof R.

18. The peptide of any one of the preceding embodiments, wherein thepeptide comprises at least one residue of R₄, R₅, R₆, R₇, R₈, S₄, S₅,S₆, S₇, or S₈.

19. The peptide of any one of the preceding embodiments, wherein thepeptide comprises at least one residue of M_(A), M_(B), M_(C), M_(D),M_(E), M_(F), M_(G), M_(H), or M_(I).

20. The peptide of any one of the preceding embodiments, wherein a sidechain of X³ and a side chain of X¹⁰ are taken together to form a staple.

21. The peptide of any one of the preceding embodiments, wherein X¹ is aresidue of an amino acid selected from P, A, D, E, F, G, H, I, K, L, M,N, Q, R, S, T, V, W, Y, and α-methyl proline.

22. The peptide of any one of the preceding embodiments, wherein X¹ is aresidue of an amino acid selected from P, A, D, E, F, G, H, I, K, L, M,N, Q, R, S, T, V, W, and Y.

23. The peptide of embodiment 21, wherein X¹ is a residue of an aminoacid selected from P, K, N, Q, R, Y, and α-methyl proline.

24. The peptide of embodiment 21, wherein X¹ is a residue of an aminoacid P.

25. The peptide of any one of the preceding embodiments, wherein X² is aresidue of an amino acid selected from A, D, E, F, G, H, I, K, L, M, N,P, Q, R, S, T, V, W, and Y.

26. The peptide of embodiment 25, wherein X² is a residue of an aminoacid selected from A, D, E, K, N, Q, and R.

27. The peptide of embodiment 25, wherein X² is a residue of A.

28. The peptide of any one of the preceding embodiments, wherein X⁴ is aresidue of an amino acid selected from I, F, H, L, V,homoleucine,tert-leucine, 3-cyclopropylalanine, 3-cyclobutylalanine,3-cyclopentylalanine, 3-cyclohexylalanine, and alpha-neopentylglycine.29. The peptide of any one of the preceding embodiments, wherein X⁴ is aresidue of an amino acid selected from I, F, H, L, and V.30. The peptide of embodiment 28, wherein X⁴ is a residue of an aminoacid selected from I, L, V, homoleucine, tert-leucine,3-cyclopropylalanine, 3-cyclobutylalanine, 3-cyclopentylalanine, andalpha-neopentylglycine.31. The peptide of embodiment 28, wherein X⁴ is a residue of I.32. The peptide of any one of the preceding embodiments, wherein X⁵ is aresidue of an amino acid selected from L, F, H, I, V, alpha-methylleucine, homoleucine, tert-leucine, 3-cyclopropylalanine,3-cyclobutylalanine, 3-cyclopentylalanine, 3-cyclohexylalanine, andalpha-neopentylglycine.33. The peptide of any one of the preceding embodiments, wherein X⁵ is aresidue of an amino acid selected from L, F, H, I, and V.34. The peptide of embodiment 32, wherein X⁵ is a residue of an aminoacid selected from L, I, V, alpha-methyl leucine, homoleucine,tert-leucine, 3-cyclopropylalanine, 3-cyclobutylalanine,3-cyclopentylalanine, 3-cyclohexylalanine, and alpha-neopentylglycine.35. The peptide of embodiment 32, wherein X⁵ is a residue of L.36. The peptide of any one of the preceding embodiments, wherein X⁶ is aresidue of an amino acid selected from D, A, E, F, H, I, K, L, M, N, P,Q, R, S, T, V, W, Y, methionine sulfone, 2-aminoadipic acid, asparticacid beta-methylester, aspartic acid beta-cyclohexylester, aspartic acidbeta-benzylester, glutamic acid beta-methylester, glutamic acidbeta-cyclohexylester, and glutamic acid beta-benzyl ester.37. The peptide of any one of the preceding embodiments, wherein X⁶ is aresidue of an amino acid selected from D, A, E, F, H, I, K, L, M, N, P,Q, R, S, T, V, W, and Y.38. The peptide of embodiment 36, wherein X⁶ is a residue of an aminoacid selected from D, E, H, N, Q, S, T, Y, methionine sulfone,2-aminoadipic acid, aspartic acid beta-methylester, aspartic acidbeta-cyclohexylester, aspartic acid beta-benzylester, glutamic acidbeta-methylester, glutamic acid beta-cyclohexylester, and glutamic acidbeta-benzyl ester.39. The peptide of embodiment 36, wherein X⁶ is a residue of an aminoacid selected from D, N, and T.40. The peptide of any one of the preceding embodiments, wherein X⁷ is aresidue of an amino acid selected from R₄, R₅, R₆, R₇, R₈, S₄, S₅, S₆,S₇, S₈, M_(A), M_(B), M_(C), M_(D), M_(E), M_(F), M_(G), M_(H), M_(I),A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y and alpha-methylalanine.41. The peptide of any one of the preceding embodiments, wherein X⁷ is aresidue of an amino acid selected from A, D, E, F, H, I, K, L, M, N, P,Q, R, S, T, V, W, Y and alpha-methyl alanine.42. The peptide of embodiment 40, wherein X⁷ is a residue of an aminoacid selected from R₄, R₅, R₆, R₇, R₈, S₄, S₅, S₆, S₇, S₈, M_(A), M_(B),M_(C), M_(D), M_(E), M_(F), M_(G), M_(H), and M_(I).43. The peptide of embodiment 40, wherein X⁷ is a residue of an aminoacid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W,and Y.44. The peptide of embodiment 40, wherein X⁷ is a residue of an aminoacid selected from A, D, E, I, K, L, N, Q, R, S, T, V, W, Y andalpha-methyl alanine.45. The peptide of embodiment 40, wherein X⁷ is a A or alpha-methylalanine residue.46. The peptide of any one of the preceding embodiments, wherein X⁸ is aresidue of an amino acid selected from H, F, I, L, N, Q, V,1-methylhistidine, 3-methylhistidine, 3-(2-pyridyl)alanine,3-(3-pyridyl)alanine, 3-(4-pyridyl)alanine, beta-2-furylalanine,beta-2-thienylalanine, 3-(2-tetrazolyl)alanine), andbeta-4-thiazolylalanine.47. The peptide of any one of the preceding embodiments, wherein X⁸ is aresidue of an amino acid selected from H, F, I, L, N, Q, and V.48. The peptide of embodiment 46, wherein X⁸ is a residue of an aminoacid selected from H, N, Q, 1-methylhistidine, 3-methylhistidine,3-(2-pyridyl)alanine, 3-(3-pyridyl)alanine, 3-(4-pyridyl)alanine,beta-2-furylalanine, beta-2-thienylalanine, 3-(2-tetrazolyl)alanine),and beta-4-thiazolylalanine.49. The peptide of embodiment 46, wherein X⁸ is a H residue.50. The peptide of any one of the preceding embodiments, wherein X⁹ is aresidue of an amino acid selected from I, V, F, H, L, homoleucine,tert-leucine, 3-cyclopropylalanine, 3-cyclobutylalanine,3-cyclopentylalanine, 3-cyclohexylalanine, and alpha-neopentylglycine.51. The peptide of any one of the preceding embodiments, wherein X⁹ is aresidue of an amino acid selected from I, V, F, H, and L.52. The peptide of embodiment 50, wherein X⁹ is a residue of an aminoacid selected from I, V, L, homoleucine, tert-leucine,3-cyclopropylalanine, 3-cyclobutylalanine, 3-cyclopentylalanine,3-cyclohexylalanine, and alpha-neopentylglycine.53. The peptide of embodiment 50, wherein X⁹ is a residue of an aminoacid selected from I and V.54. The peptide of any one of the preceding embodiments, wherein X¹¹ isa residue of an amino acid selected from R, A, D, E, F, H, I, K, L, M,N, P, Q, S, T, V, W, Y, 3-(1-naphthylalanine), 2-aminoadipic acid,asymmetric dimethylarginine, symmetric dimethylarginine, homoarginine,N-epsilon-methyllysine, N-epsilon-dimethyllysine, andN-epsilon-trimethyllysine.55. The peptide of any one of the preceding embodiments, wherein X¹¹ isa residue of an amino acid selected from R, A, D, E, F, H, I, K, L, M,N, P, Q, S, T, V, W, and Y.56. The peptide of embodiment 54, wherein X¹¹ is a residue of an aminoacid selected from R, A, E, F, K, Q, S, V, Y, 3-(1-naphthylalanine),2-aminoadipic acid, asymmetric dimethylarginine, symmetricdimethylarginine, homoarginine, N-epsilon-methyllysine,N-epsilon-dimethyllysine, and N-epsilon-trimethyllysine.57. The peptide of embodiment 54, wherein X¹¹ is a residue of an aminoacid selected from R, A, F, K, S, V, 3-(1-naphthylalanine), asymmetricdimethylarginine, symmetric dimethylarginine, homoarginine, andN-epsilon-methyllysine.58. The peptide of any one of the preceding embodiments, wherein X¹² isa residue of an amino acid selected from V, F, H, I, L, alpha-methylvaline, alpha methyl leucine, homoleucine, tert-leucine,3-cyclopropylalanine, 3-cyclobutylalanine, 3-cyclopentylalanine,3-cyclohexylalanine, and alpha-neopentylglycine.59. The peptide of any one of the preceding embodiments, wherein X¹² isa residue of an amino acid selected from V, F, H, I, and L.60. The peptide of embodiment 58, wherein X¹² is a residue of an aminoacid selected from I, A, L, V, alpha-methylleucine, homoleucine,tert-leucine, 3-cyclopropylalanine, 3-cyclobutylalanine,3-cyclopentylalanine, 3-cyclohexylalanine, alpha-neopentylglycine,O-propargylserine, L-octylglycine, and L-alloisoleucine.61. The peptide of embodiment 58, wherein X¹² is a residue of an aminoacid selected from V, alpha-methyl valine, and alpha methyl leucine.62. The peptide of any one of the preceding embodiments, wherein X¹³ isa residue of an amino acid selected from W, A, D, E, F, H, I, K, L, M,N, P, Q, R, S, T, V, Y, d-tryptophan, alpha-methyl tryptophan,3-(1-naphthylalanine), 3-(2-naphthylalanine), 4-chlorotryptophan,5-chlorotryptophan, 6-chlorotryptophan, 7-chlorotryptophan,4-bromotryptophan, 5-bromotryptophan, 6-bromotryptophan,7-bromotryptophan, 4-fluorotryptophan, 5-fluorotryptophan,6-fluorotryptophan, 7-fluorotryptophan, 1-methyltryptophan,2-methyltryptophan, 4-methyltryptophan, 5-methyltryptophan,6-methyltryptophan, 7-methyltryptophan, 2-hydroxytryptophan,4-hydroxytryptophan, 5-hydroxytryptophan, 6-hydroxytryptophan,7-hydroxytryptophan, 5-methoxytryptophan, 7-azatryptophan,3-benzothienylalanine, and 4-phenyl-L-phenylalanine.63. The peptide of any one of the preceding embodiments, wherein X¹³ isa residue of an amino acid selected from W, A, D, E, F, H, I, K, L, M,N, P, Q, R, S, T, V, and Y.64. The peptide of embodiment 62, wherein X¹³ is a residue of an aminoacid selected from W, D, E, F, Y, d-tryptophan, alpha-methyl tryptophan,3-(1-naphthylalanine), 3-(2-naphthylalanine), 5-chlorotryptophan,6-chlorotryptophan, 7-chlorotryptophan, 5-bromotryptophan,6-bromotryptophan, 7-bromotryptophan, 5-fluorotryptophan,6-fluorotryptophan, 7-fluorotryptophan, 1-methyltryptophan,2-methyltryptophan, 5-methyltryptophan, 6-methyltryptophan,7-methyltryptophan, 2-hydroxytryptophan, 5-hydroxytryptophan,6-hydroxytryptophan, 7-hydroxytryptophan, 5-methoxytryptophan,7-azatryptophan, and 3-benzothienylalanine.65. The peptide of embodiment 62, wherein X¹³ is a residue of an aminoacid selected from W, D-tryptophan, and alpha-methyl tryptophan.66. The peptide of any one of the preceding embodiments, wherein thepeptide comprising a (i, i+4) staple wherein, not including the twoamino acid residues that are directly connected to the staple, there arethree amino acid residues between the two amino acid residues that aredirectly connected to the staple.67. The peptide of any one of the preceding embodiments, wherein thepeptide comprising a (i, i+7) staple wherein, not including the twoamino acid residues that are directly connected to the staple, there aresix amino acid residues between the two amino acid residues that aredirectly connected to the staple.68. The peptide of any one of the preceding embodiments, wherein thestaple is formed by olefin metathesis of two terminal olefins each ofwhich is independently of a side chain of an amino acid residue.69. The peptide of any one of the preceding embodiments, wherein thestaple is formed by olefin metathesis of two terminal olefins each ofwhich is independently of a side chain of a residue of an amino acidselected from R₄, R₅, R₆, R₇, R₈, S₄, S₅, S₆, S₇, S₈, M_(A), M_(B),M_(C), M_(D), M_(E), M_(F), M_(G), M_(H), and M_(I).70. The peptide of any one of the preceding embodiments, wherein thepeptide comprises one and no more than one staple.71. The peptide of any one of the preceding embodiments, wherein thepeptide comprises two or more staples.72. The peptide of embodiment 71, wherein at least two staples arebonded to the same peptide backbone atom.73. The peptide of embodiment 71, wherein none of the staples are bondedto the same peptide backbone atom.74. The peptide of any one of the preceding embodiments, wherein astaple is bonded to a peptide backbone atom of amino acid residue 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17.75. The peptide of any one of the preceding embodiments, wherein astaple is L^(s), wherein L^(s)is an optionally substituted, bivalentC₁₋₅₀ aliphatic group wherein one or more methylene units of thealiphatic group are optionally and independently replaced with —C(R′)₂—,-Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—,or —C(O)O—;

each -Cy- is independently an optionally substituted bivalent groupselected from a C₃₋₂₀ cycloaliphatic ring, a C₆₋₂₀ aryl ring, a 5-20membered heteroaryl ring having 1-10 heteroatoms independently selectedfrom oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20membered heterocyclyl ring having 1-10 heteroatoms independentlyselected from oxygen, nitrogen, sulfur, phosphorus and silicon;

each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R;

each R is independently —H, or an optionally substituted group selectedfrom C₁₋₃₀ aliphatic, C₁₋₃₀ heteroaliphatic having 1-10 heteroatomsindependently selected from oxygen, nitrogen, sulfur, phosphorus andsilicon, C₆₋₃₀ aryl, C₆₋₃₀ arylaliphatic, C₆₋₃₀ arylheteroaliphatichaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon, or

two R groups are optionally and independently taken together to form acovalent bond, or:

two or more R groups on the same atom are optionally and independentlytaken together with the atom to form an optionally substituted, 3-30membered, monocyclic, bicyclic or polycyclic ring having, in addition tothe atom, 0-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon; or

two or more R groups on two or more atoms are optionally andindependently taken together with their intervening atoms to form anoptionally substituted, 3-30 membered, monocyclic, bicyclic orpolycyclic ring having, in addition to the intervening atoms, 0-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon.

76. A peptide having the structure of:

or a salt thereof, wherein

each of R^(a), R¹, R², R³, and R⁴ is independently R′;

R^(b) is R′, —OR′ or —N(R′)₂;

each of X is independently an amino acid residue;

each of a, b, c, s, and d is independently 1-20;

each of C¹ and C² is independently a carbon atom;

each L^(s) is independently -L^(s1)-L^(s2)-L^(s3)-, wherein L^(s1) isbonded to C¹ and L^(s3) is bonded to C²;

each of L^(s1), L^(s2), and L^(s3) is independently L;

each L is independently a covalent bond, or an optionally substituted,bivalent C₁-C₂₀ aliphatic group wherein one or more methylene units ofthe aliphatic group are optionally and independently replaced with—C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—,—S(O)₂N(R′)—, —C(O)S—, or —C(O)O—;

each -Cy- is independently an optionally substituted bivalent groupselected from a C₃₋₂₀ cycloaliphatic ring, a C₆₋₂₀ aryl ring, a 5-20membered heteroaryl ring having 1-10 heteroatoms independently selectedfrom oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20membered heterocyclyl ring having 1-10 heteroatoms independentlyselected from oxygen, nitrogen, sulfur, phosphorus and silicon;

each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R;

each R is independently —H, or an optionally substituted group selectedfrom C₁₋₃₀ aliphatic, C₁₋₃₀ heteroaliphatic having 1-10 heteroatomsindependently selected from oxygen, nitrogen, sulfur, phosphorus andsilicon, C₆₋₃₀ aryl, C₆₋₃₀ arylaliphatic, C₆₋₃₀ arylheteroaliphatichaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon, or

two R groups are optionally and independently taken together to form acovalent bond, or:

two or more R groups on the same atom are optionally and independentlytaken together with the atom to form an optionally substituted, 3-30membered, monocyclic, bicyclic or polycyclic ring having, in addition tothe atom, 0-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon; or

two or more R groups on two or more atoms are optionally andindependently taken together with their intervening atoms to form anoptionally substituted, 3-30 membered, monocyclic, bicyclic orpolycyclic ring having, in addition to the intervening atoms, 0-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon.

77. The peptide of embodiment 76, wherein the sum of all a, b, c, and dis 5 to 70.

78. The peptide of embodiment 77, wherein the sum of all a, b, c, and dis 10 to 20.

79. The peptide of any one of embodiments 76-78, wherein a is 1-20.

80. The peptide of any one of embodiments 76-79, wherein b is 2-6.

81. The peptide of any one of embodiments 76-80, wherein c is 1-20.

82. The peptide of any one of embodiments 76-81, wherein d is 1-20.

83. The peptide of any one of embodiments 76-82, wherein the peptide isa peptide of any one of embodiments 1-74.

84. A stapled peptide comprising a staple having the structure of L^(s),wherein:

L^(s) is -L^(s1)-L^(s2)-L^(s3)-;

each of L^(s1), L^(s2), and L^(s3) is independently L;

each L is independently a covalent bond, or an optionally substituted,bivalent C₁-C₂₀ aliphatic group wherein one or more methylene units ofthe aliphatic group are optionally and independently replaced with—C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—,—C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—,—S(O)₂N(R′)—, —C(O)S—, or —C(O)O—;

each -Cy- is independently an optionally substituted bivalent groupselected from a C₃₋₂₀ cycloaliphatic ring, a C₆₋₂₀ aryl ring, a 5-20membered heteroaryl ring having 1-10 heteroatoms independently selectedfrom oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20membered heterocyclyl ring having 1-10 heteroatoms independentlyselected from oxygen, nitrogen, sulfur, phosphorus and silicon;

each R′ is independently —R, —C(O)R, —CO₂R, or —SO₂R;

each R is independently —H, or an optionally substituted group selectedfrom C₁₋₃₀ aliphatic, C₁₋₃₀ heteroaliphatic having 1-10 heteroatomsindependently selected from oxygen, nitrogen, sulfur, phosphorus andsilicon, C₆₋₃₀ aryl, C₆₋₃₀ arylaliphatic, C₆₋₃₀ arylheteroaliphatichaving 1-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon, or

two R groups are optionally and independently taken together to form acovalent bond, or:

two or more R groups on the same atom are optionally and independentlytaken together with the atom to form an optionally substituted, 3-30membered, monocyclic, bicyclic or polycyclic ring having, in addition tothe atom, 0-10 heteroatoms independently selected from oxygen, nitrogen,sulfur, phosphorus and silicon; or

two or more R groups on two or more atoms are optionally andindependently taken together with their intervening atoms to form anoptionally substituted, 3-30 membered, monocyclic, bicyclic orpolycyclic ring having, in addition to the intervening atoms, 0-10heteroatoms independently selected from oxygen, nitrogen, sulfur,phosphorus and silicon.

85. The peptide of embodiment 84, wherein:

one end of L^(s) is connected to an atom A^(n1) of the peptide backbone,wherein A^(n1) is bonded to R¹;

one end of L^(s) is connected to an atom A^(n2) of the peptide backbone,wherein A² is bonded to R²;

each of R¹ and R² is independently R′;

there are m amino acid residues between the amino acid residuecomprising A^(n1) and the amino acid residue comprising A^(n2), notincluding the amino acid residue comprising A^(n1) and the amino acidresidue comprising A^(n2); and

m is an integer of 1-12.

86. The peptide of embodiment 85, wherein each of A^(n1) and A² isindependently a carbon atom.

87. The peptide of embodiment 85, wherein each of A^(n1) and A² isindependently an alpha carbon atom.

88. The peptide of any one of embodiments 85-87, wherein m is 1.

89. The peptide of any one of embodiments 85-87, wherein m is 2.

90. The peptide of any one of embodiments 85-87, wherein m is 3.

91. The peptide of any one of embodiments 85-87, wherein m is 4.

92. The peptide of any one of embodiments 85-87, wherein m is 5.

93. The peptide of any one of embodiments 85-87, wherein m is 6.

94. The peptide of any one of embodiments 85-87, wherein m is 7.

95. The peptide of any one of embodiments 76-94, wherein C¹ or A^(n1)has an R configuration.

96. The peptide of any one of embodiments 76-94, wherein C¹ or A^(n1)has an S configuration.

97. The peptide of any one of embodiments 76-94, wherein C¹ or A^(n1) isachiral.

98. The peptide of any one of embodiments 76-97, wherein C² or A^(n2)has an R configuration.

99. The peptide of any one of embodiments 76-97, wherein C² or A² has anS configuration.

100. The peptide of any one of embodiments 76-97, wherein C² or A² isachiral.

101. The peptide of any one of embodiments 76-97, wherein C² or A² isachiral.

102. The peptide of any one of the preceding embodiments, wherein astaple is L^(s), wherein L^(s) is an optionally substituted, bivalentC₈₋₁₄ aliphatic group wherein one or more methylene units of thealiphatic group are optionally and independently replaced with —C(R′)₂—,-Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—,or —C(O)O—.103. The peptide of any one of the preceding embodiments, wherein astaple is L^(s), wherein L^(s) is an optionally substituted, bivalentC₉₋₁₃ aliphatic group wherein one or more methylene units of thealiphatic group are optionally and independently replaced with —C(R′)₂—,-Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—,or —C(O)O—.104. The peptide of any one of the preceding embodiments, wherein astaple is L^(s), wherein L^(s) is an optionally substituted, bivalentC₁₀₋₁₅ aliphatic group wherein one or more methylene units of thealiphatic group are optionally and independently replaced with —C(R′)₂—,-Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—,or —C(O)O—.105. The peptide of any one of the preceding embodiments, wherein astaple is L^(s), wherein L^(s)is an optionally substituted, bivalentC₁₁₋₁₄ aliphatic group wherein one or more methylene units of thealiphatic group are optionally and independently replaced with —C(R′)₂—,-Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(O)O—, —S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—,or —C(O)O—.106. The peptide of any one of embodiments 102-105, wherein the stapleis a (i, i+4) staple.107. The peptide of any one of embodiments 102-105, wherein the stapleis a (i, i+7) staple.108. The peptide of any one of the preceding embodiments, wherein foreach of L^(s), L^(s1), L^(s2) and L^(s3), any replacement of methyleneunits, if any, is replaced with —N(R′)— or —N(R′)—C(O)—.109. The peptide of any one of the preceding embodiments, wherein astaple is a hydrocarbon staple.110. The peptide of embodiment 109, wherein the hydrocarbon staple isL^(s), wherein L^(s) is C₅₋₂₀ bivalent aliphatic.111. The peptide of any one of embodiments 1-108, wherein a staplecomprises a —N(R′)-moiety.112. The peptide of any one of embodiments 1-108, wherein a staplecomprises a —N(R′)-moiety, wherein the —N(R′)— moiety is not bonded to acarbon atom that also forms a double bond with a heteroatom.113. The peptide of any one of embodiments 1-108, wherein a staplecomprises a —N(R′)-moiety, wherein the —N(R′)— moiety is not bonded to—C(O)—.114. The peptide of any one of embodiments 1-108, wherein a staplecomprises a —N(R′)—C(O)— moiety.115. The peptide of any one of embodiments 75-108, wherein at least onemethylene unit is replaced with —(NR′)—.116. The peptide of any one of embodiments 75-108, wherein at least onemethylene unit is replaced with —(NR′)—, wherein the —N(R′)— moiety isnot bonded to —C(O)—.117. The peptide of any one of embodiments 75-108, wherein at least onemethylene unit is replaced with —(NR′)—C(O)—.118. The peptide of any one of embodiments 111-117, wherein R′ of the—N(R′)— is R.119. The peptide of any one of embodiments 111-117, wherein R′ of the—N(R′)— is optionally substituted C₁₋₆ alkyl.120. The peptide of any one of embodiments 111-117, wherein R′ of the—N(R′)— is methyl.121. The peptide of any one of embodiments 75-120, wherein L^(s1)comprises at least one methylene units replaced with —N(R′)—.122. The peptide of any one of embodiments 75-120, wherein L^(s1)comprises at least one methylene units replaced with —N(R′)—, whereinnone of the neighboring methylene unit is replaced with —C(O)—.123. The peptide of any one of embodiments 75-120, wherein L^(s1)comprises at least one —N(R′)C(O)O—.124. The peptide of any one of embodiments 75-120, wherein L^(s1) is-L′-N(R′)—.125. The peptide of any one of embodiments 75-120, wherein L^(s1) is-L′-N(R′)C(O)O—.126. The peptide of any one of embodiments 124-125, wherein L′ is C₁₋₆alkylene.127. The peptide of any one of embodiments 124-126, wherein L′ is bondedto a peptide backbone atom.128. The peptide of any one of embodiments 75-120, wherein L^(s1) isoptionally substituted C₁₋₁₀ bivalent aliphatic.129. The peptide of any one of embodiments 75-120, wherein L^(s1) isoptionally substituted C₁₋₁₀ bivalent alkylene.130. The peptide of any one of embodiments 75-126, wherein L^(s2) isoptionally substituted bivalent C₁-C₆ aliphatic.131. The peptide of any one of embodiments 75-126, wherein L^(s2) is—CH₂—CH═CH—CH₂—.132. The peptide of any one of embodiments 75-126, wherein L^(s2) is-(E)-CH₂—CH═CH—CH₂—.133. The peptide of any one of embodiments 75-126, wherein L^(s2) is—(Z)—CH₂—CH═CH—CH₂—.134. The peptide of any one of embodiments 75-126, wherein L^(s2) is—(CH₂)₄—.135. The peptide of any one of embodiments 75-134, wherein L^(s3)comprises at least one methylene units replaced with —N(R′)—.136. The peptide of any one of embodiments 75-134, wherein L^(s3)comprises at least one methylene units replaced with —N(R′)—, whereinnone of the neighboring methylene unit is replaced with —C(O)—.137. The peptide of any one of embodiments 75-134, wherein L^(s3)comprises at least one —N(R′)C(O)O—.138. The peptide of any one of embodiments 75-134, wherein L^(s3) is-L′-N(R′)—.139. The peptide of any one of embodiments 75-134, wherein L^(s3) is-L′-N(R′)C(O)O—.140. The peptide of any one of embodiments 138-139, wherein L′ is C₁₋₆alkylene.141. The peptide of any one of embodiments 138-140, wherein L′ is bondedto a peptide backbone atom.142. The peptide of any one of embodiments 75-134, wherein L^(s3) isoptionally substituted C₁₋₁₀ bivalent aliphatic.143. The peptide of any one of embodiments 75-134, wherein L^(s3) isoptionally substituted C₁₋₁₀ bivalent alkylene.144. The peptide of any one of embodiments 75-134, wherein L^(s3) isoptionally substituted C₁₋₁₀ bivalent alkylene.145. The peptide of any one of embodiments 75-134, wherein L^(s) is astaple of Table S-1.146. The peptide of any one of embodiments 75-134, wherein L^(s) is astaple of Table S-2.147. The peptide of any one of embodiments 75-134, wherein L^(s) is astaple of Table S-3.148. The peptide of any one of embodiments 75-134, wherein L^(s) is astaple of Table S-4.149. The peptide of any one of the preceding embodiments, wherein astaple has 5-20 staple chain atoms, wherein the chain of the staple isthe shortest covalent connection in the staple from a first end of astaple to a second end of the staple, wherein the first end and thesecond end connect to different peptide backbone atoms.150. The peptide of embodiment 149, wherein a staple has 8 staple chainatoms.151. The peptide of embodiment 149, wherein a staple has 9 staple chainatoms.152. The peptide of embodiment 149, wherein a staple has 10 staple chainatoms.153. The peptide of embodiment 149, wherein a staple has 11 staple chainatoms.154. The peptide of embodiment 149, wherein a staple has 12 staple chainatoms.155. The peptide of embodiment 149, wherein a staple has 13 staple chainatoms.156. The peptide of embodiment 149, wherein a staple has 14 staple chainatoms.157. The peptide of embodiment 149, wherein a staple has 15 staple chainatoms.158. The peptide of embodiment 149, wherein a staple has 16 staple chainatoms.159. The peptide of any one of embodiments 1-74, wherein the peptide isa peptide of any one of embodiments 75-158.160. The peptide of any one of the preceding embodiments, wherein thepeptide has a sequence that is at least 50%, 60%, 70%, 80%, 90%, or 95%homology with a peptide of Table 1.161. The peptide of any one of the preceding embodiments, wherein thepeptide is a peptide of Table 1.162. The peptide of any one of the preceding embodiments, wherein thepeptide can form a helix structure.163. The peptide of any one of the preceding embodiments, wherein thepeptide has a solubility of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, or 250 uM in DPBS (per liter, 8 g sodiumchloride, 0.2 g potassium phosphate, monobasic, 1.15 g sodium phosphate,dibasic, and 0.2 g potassium chloride).164. The peptide of embodiment 163, wherein the solubility is at least 1uM.165. The peptide of embodiment 163, wherein the solubility is at least 5uM.166. The peptide of embodiment 163, wherein the solubility is at least10 uM.167. The peptide of embodiment 163, wherein the solubility is at least50 uM.168. The peptide of embodiment 163, wherein the solubility is at least100 uM.169. The peptide of embodiment 163, wherein the solubility is at least200 uM.170. The peptide of any one of the preceding embodiments, wherein thepeptide binds to beta-catenin.171. The peptide of any one of the preceding embodiments, wherein thepeptide has a Kd of no greater than 1, 2, 3, 4, 5, or 10 uM forbeta-catenin.172. The peptide of any one of the preceding embodiments, wherein thepeptide has a Kd of no greater than 1 uM for beta-catenin.173. The peptide of any one of embodiments 171-172, wherein the Kd ismeasured by fluorescence polarization, surface plasmon resonance, orTR-FRET.174. The peptide of any one of the preceding embodiments, wherein thepeptide has less than 10%, 15%, 20%, 25%, 30%, 40%, 50% non-specificcytotoxicity at a concentration of no less than 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 uM as measured by a LDH release assay compared to an appreciatepositive reference.175. The peptide of any one of the preceding embodiments, wherein thepeptide comprises a staple comprising a —N(R′)— or —N(R′)—C(O)— moiety,and has lower non-specific cytotoxicity compared to a peptide comprisinga hydrocarbon staple but is otherwise of the identical structure whenassayed under a comparable condition.176. The peptide of any one of the preceding embodiments, wherein thepeptide binds to beta-catenin selectively at sites that interact withAxin over sites that interacts with BCL9.177. The peptide of any one of the preceding embodiments, wherein thepeptide binds to beta-catenin selectively at sites that interact withAxin over sites that interacts with BCL9 as measured in an appropriatecompetition fluorescence polarization assay.178. The peptide of any one of the preceding embodiments, wherein thepeptide is conjugated with a second entity.179. The peptide of embodiment 178, wherein the second entity is alabel.180. The peptide of embodiment 178, wherein the second entity a labelselected from biotin and a fluorescence label.181. The peptide of embodiment 178, wherein the second entity is atargeting moiety.182. The peptide of embodiment 178 or 181, wherein the second entity isa carbohydrate moiety.183. The peptide of embodiment 182, wherein the second entity is orcomprises a GalNac moiety.184. The peptide of embodiment 178, wherein the second entity is a lipidmoiety.185. The peptide of any one of the preceding embodiments, wherein eachamino acid residue is independently a residue of an amino acid offormula A-I, A-II or A-III.186. A pharmaceutical composition comprising a peptide of any one of thepreceding embodiments and pharmaceutically acceptable carrier.187. A method for modulating a function of beta-catenin, comprisingcontacting a system comprising beta-catenin with a peptide of any one ofthe preceding embodiments.188. A method for modulating a function of Wnt signaling pathway,comprising contacting a system comprising the pathway with a peptide ofany one of the preceding embodiments.189. A method for modulating interaction of beta-catenin with Axin,comprising contacting a system comprising beta-catenin with a peptide ofany one of the preceding embodiments.190. A method for modulating expression of a nucleic acid sequence in asystem, comprising contacting a system comprising beta-catenin a peptideof any one of the preceding embodiments; wherein expression of thenucleic acid sequence is associated with beta-catenin.191. A method for modulating level of a product encoded by a nucleicacid sequence in a system, comprising contacting a system comprisingbeta-catenin a peptide of any one of the preceding embodiments;wherein level of a product encoded by a nucleic acid sequence isassociated with beta-catenin.192. The peptide of embodiment 191, wherein the product is a protein.193. The peptide of embodiment 191, wherein the product is mRNA.194. A method for preventing or treating a beta-catenin-associatedcondition, disorder, or disease, comprising administering to a subjectsusceptible thereto or suffering therefrom a peptide or a composition ofany one of the preceding embodiments.195. The method of embodiment 194, wherein the condition, disorder, ordisease is cancer.196. The method of embodiment 195, wherein the cancer is colorectalcancer.197. The method of embodiment 195, wherein the cancer is hepatocellularcancer.198. The method of embodiment 195, wherein the cancer is prostatecancer.199. The method of embodiment 195, wherein the cancer is melanoma.200. The method of any one of embodiments 194-199, wherein the peptideor composition is administered prior to, concurrently with, orsubsequent to an additional agent.201. The method of embodiment 200, wherein the additional agent is ananti-cancer drug.202. The method of embodiment 200, wherein the additional agent is achemotherapy agent.203. The method of embodiment 200, wherein the additional agent is animmuno oncology drug.204. The method of embodiment 200, wherein the additional agent is acheckpoint inhibitor.205. The method of embodiment 200, wherein the additional agent is ananti-PD1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody.206. The method of embodiment 194, wherein the condition, disorder, ordisease is a heart condition, disorder, or disease.

EXEMPLIFICATION

Non-limiting examples of provided technologies are described below.Those having ordinary skill in the art appreciates that varioustechnologies can be utilized to prepare and access compounds,compositions and methods in accordance with the present disclosure.

Example 1. Exemplary Preparation of Provided Agents

Provided agents, e.g., stapled peptides, can be prepared using varioustechnologies in accordance with the present disclosure, for example,methods as described herein. As appreciated by those skilled in the art,parameters of provided methods, e.g., steps, reagents, solvents,concentrations, temperatures, time, etc., may be optimized as desired.

In some embodiments, peptides can be prepared on a peptide synthesizer.For example, in some embodiments, provided peptides were typicallysynthesized on an Intavis Multipep RSi peptide synthesizer using Fmocsolid phase peptide chemistry on CEM ProTide Rink Amide resin (loading0.55-0.8 mmol/g). In some embodiments, resin for synthesis is swelled ina suitable solvent, e.g., NMP, at a suitable temperature for a period oftime (e.g., at 45 degrees for 20 minutes in a 5 mL or 2 mL plasticfritted reaction vessel). Amino acid residues are then added usingpeptide synthesis procedures (typically at 45 degrees; conditions can beadjusted as necessary). In some embodiments, provided stapled peptides,e.g., those described in Table 1, were prepared as described below.

Peptides were typically synthesized on an Intavis Multipep RSi peptidesynthesizer using Fmoc solid phase peptide chemistry on CEM ProTide RinkAmide resin (loading 0.55-0.8 mmol/g). Resin for synthesis was swelledin NMP at 45 degrees for 20 minutes in a 5 mL or 2 mL plastic frittedreaction vessel. Amino acid residues were added using the followingprocedure (all steps at 45 degrees).

a) The Fmoc group was removed using one five-minute treatment and oneten-minute treatment with 20% Piperidine (v/v), 0.1 M HOBT in NMP.

b) The resin was washed eight times with NMP.

c) 5 equivalents of 0.5 M amino acid solution, 5 equivalents of 2 M DIC,and 5 equivalents of 0.5 M Oxyma were added to a preactivation vesselfor one minute.

d) Reaction mixture was added to the reaction vessel and coupled for 30minutes, vortexing intermittently.

e) Reaction vessel was washed once with NMP.

f) Steps c), d), and e) were repeated. In some embodiments, in the caseof difficult positions, steps c), d), and e) were repeated twice.

g) Any unreacted amines were capped with 5% (v/v) acetic anhydride inNMP for two minutes.

h) The reaction vessels were washed, e.g., eight times with NMP.

Following the final residue, the Fmoc group was removed using theprocedure from steps a) above and the peptides were typically capped (insome cases, were not capped so that the 5′-amino group can react withother entities as exemplified in the present disclosure), e.g., with 5%(v/v) acetic anhydride in NMP for 15 minutes at 45 degrees for Accapping. The resin was washed 5 times with DCM.

Staples can be formed using various technologies in accordance with thepresent disclosure. In some embodiments, staples are formed by olefinmetathesis. In some embodiments, two amino acid side chains eachindependently comprising an olefin (in some embodiments, terminalolefin) are reacted with each other under suitable olefin metathesisconditions so that olefin metathesis happens between the two side chainsand a staple is formed. Many olefin metathesis conditions (e.g.,catalyst, solvent, temperature, etc.) are known in the art and can beutilized in accordance with the present disclosure.

For example, in an exemplary procedure that was used to prepare providedstaples, e.g., those in Table 1, resin with peptides, e.g., as preparedabove, was swelled at 40 degrees in DCE for 20 minutes on the IntavisMultipep RSi. The peptides were treated with 30 mol % of a freshlyprepared 5 mM solution of bis(tricyclohexvlphosphine)benzylideneruthenium (IV) dichloride (Grubb's I) in DCE for one hour, withvortexing continuously. The treatment was repeated depending on e.g.,conversion, purity, etc. The resin was then washed 5 times with DCM. Thepeptides were cleaved from the resin and de-protected using 95%trifluoroacetic acid, 2.5% triisopropylsilane, and 2.5% water for twoand a half hours vortexing at room temperature. After TFA was evaporatedunder an inert atmosphere, e.g., nitrogen gas, the peptides wereprecipitated in a suitable solvent, e.g., tert-butyl methyl ether.

In some embodiments, Hoveyda-Grubbs catalyst may be used and may providebetter yields, purity and/or selectivity.

Peptides can be further processed as desired. For example, in someembodiments, provided stapled peptides comprising an olefin in a staplecan be subjected to a reduction (e.g., hydrogenation) condition, so thatan olefin moiety in a staple is hydrogenated and converted into analkane moiety. Described below is an exemplary procedure.

In an exemplary procedure for making FP0650rc, 100 umol FP0650c onpeptide synthesis resin was swelled in N-methyl-2-pyrrolidone (NMP) in aBiotage Alstra microwave peptide synthesizer for 5 min at a temperatureof, e.g., 50° C. The solvent was removed and 1.45 mL of 1.4 M piperidine(20 equivalents) in NMP was combined with 298 mg of2,4,6-triisopropylbenzenesulfonyl hydrazide (20 equivalents) dissolvedin 1.45 mL of NMP and subsequently added to the resin. The reaction wasallowed to proceed at 50° C. for 2 hours and the resin was then washed2× with NMP and 2× with 1,2-dichloroethane (DCE). If desired, the resinwas then treated with freshly prepared reaction solution according tothe above steps (in some cases, two or more additional times) until thereaction was complete (as monitored by LC/MS after analytical cleavageof a few beads of resin). After the reaction was complete, FP0650rc wascleaved from the resin and purified.

In some embodiments, provided stapled peptides comprises one or moreamino staples which comprises an amino moiety (e.g., —N(R′)—, wherein R′is as described in the present disclosure, and the —N(R′)— is not bondedto —C(O)— groups). In some embodiments, a staple comprising an aminomoiety is prepared from extraction of CO₂ from an appropriate staplecomprising a corresponding carbamate moiety (e.g., converting—N(R′)—C(O)—O— to —N(R′)—). An exemplary procedure for preparingpeptides comprising an amino staple (e.g., those in Table 1) through,e.g., CO₂ extrusion, is described below.

Stapled peptides comprising carbamate staples were treated with 80 mol %of freshly prepared 10 mM solution of tetrakis(triphenylphosphine)palladium (0) in DCM for 90 minutes, vortexing continuously. The resinwas washed 5 times in DCM and further modifications and/or cleavage andpurification were performed using standard procedures.

As described in the present disclosure, in some embodiments, providedpeptides may be further modified, e.g., conjugated with a second entity.In some embodiments, a modification, e.g., conjugate, is at or through aN-terminus. An exemplary procedure for preparing N-terminal for furthermodification (e.g., conjugation) is described below.

Prior to final Fmoc deprotection and capping with acetic anhydride, theFmoc protected peptides were stapled via standard protocol, e.g., aprotocol described above. Following metathesis, the resin was washedwith NMP several times. The resin was swelled in NMP for 20 minutes, andtreated four times with 20% piperidine and 0.1 M HOBT in NMP for fiveminutes each at room temperature. The resin was then washed five timeswith NMP.

In some embodiments, a peptide is conjugated with biotin. An exemplaryprocedure for preparing biotinylated peptides, e.g., those in Table 1,is described below.

To prepare biotinylated peptides, a free N-terminus was coupled tobiotin with 3 equivalents of biotin (0.5 M in NMP), 3 equivalents ofCOMU (0.5 M in NMP), and 6 equivalents of DIEA (2 M in NMP) for 1 hourat room temperature (standard coupling conditions on the synthesizerproduced similar results). Cleavage and purification was then performedusing the standard procedures.

In some embodiments, a peptide is conjugated to a label, e.g., afluorescent label. An exemplary procedure for preparing such peptides,e.g., those in Table 1, is described below.

To prepare fluorescein-conjugated peptides, a free N-terminus wascoupled to FITC using five equivalents of FITC (75 mM finalconcentration) and 10 equivalents of DIEA (neat) dissolved in NMP for 10hours, vortexing continuously. Cleavage and purification was thenperformed using the standard procedures.

In some embodiments, a peptide is conjugated to an entity comprisingPEG. An exemplary procedure for preparing such peptides, e.g., those inTable 1, is described below.

To prepare peptides comprising a PEG moiety, e.g., PEG containing a freeamine handle, N-terminal Fmoc protected PEG was coupled to the stapledpeptide on resin using standard coupling conditions and then theN-terminal Fmoc was removed using standard conditions. Cleavage andpurification was then performed using standard procedures.

In some embodiments, provided compounds are purified so that a higherpurity is achieved. Various purification technologies can be utilized inaccordance with the present disclosure. In some embodiments,purification comprises one or more steps using HPLC or UPLC. In someembodiments, provided compounds, e.g., stapled peptides, where dissolvedin a small volume of a solvent, e.g., DMSO, and were purified by reversephase HPLC using a suitable column (e.g., a Rx-C8 column (Agilent)) withsuitable mobile phase conditions. Provided compounds and compositionscan be characterized using a number of technologies in accordance withthe present disclosure. In some embodiments, provided compounds werecharacterized by mass spectrometry under suitable conditions (e.g.,electrospray in positive ion mode). For example, in some embodiments,provided stapled peptides were dissolved in small volume of DMSO andwere purified by reverse phase HPLC using a Rx-C8 column (Agilent) and agradient of Acetonitrile with 0.1% TFA and Water with 0.1% TFA. HPLCfractions were characterized by LC-MS using electrospray (e.g., inpositive ion mode), pooled, and lyophilized to provide products havingthe correct characterization data (e.g., MS). Exemplary provided stapledpeptides were presented below, e.g., Table 3.

Example 2. Provided Agents Bind to Beta-Catenin

Among other things, provided agents, e.g., stapled peptides, interactwith beta-catenin and modulate its functions as demonstrated in thepresent disclosure. Various technologies are known in the art and can beutilized to assess interactions in accordance with the presentdisclosure.

In some embodiments, a direct fluorescence polarization assay is used toassess binding of provided compounds to beta-catenin. In an exemplaryprocedure, beta-catenin solutions are prepared in a buffer using serialdilution, for example, in some cases, beta-catenin solutions wereprepared in a buffer (50 mM Tris pH 8.0, 250 mM NaCl, 2% glycerol, 0.5mM EDTA, 0.02% w/v sodium azide) using a 3-fold serial dilution from 5μM. Probe solution (20 nM 5FAM or FITC labeled peptide in buffer) wasprepared and 40 μL per well plated in a black polystyrene 384-well plate(Corning). Equal volume of the serial diluted beta-catenin was added tothe plate and incubated protected from light for 15 minutes prior toread. Reads were performed on a Spectramax M5 (Molecular Devices) induplicate.

In some embodiments, a competition fluorescence polarization assay isused to assess binding of provided compounds to beta-catenin. In anexemplary procedure, solutions of provided compounds, e.g., providedstapled peptides, were prepared in a buffer (e.g., 50 mM Tris pH 8.0,250 mM NaCl, 2% glycerol, 0.5 mM EDTA, 0.02% w/v sodium azide) using a3-fold serial dilution from 5 μM. Probe solution (15 nM full-lengthß-Catenin, 20 nM FITC labeled peptide in buffer) was prepared andincubated for a period of time, e.g., 5 minutes, and the a volume, e.g.,40 μL per well plated in suitable plate, e.g., a black polystyrene384-well plate (Corning). Equal volume of the serial diluted peptide wasadded to the plate and incubated protected from light for 15 minutesprior to read. Reads were performed on a Spectramax M5 (MolecularDevices) in duplicate. Suitable probe was FITC-PEG1-PQ-S5-ILD-S5-HVRRVWR(SEQ ID NO: 1) (hydrocarbon staple formed by two S5 via olefinmetathesis) and/or FITC-bA-PQ-S5-ILD-S5-HVRRVWR (SEQ ID NO: 3)(hydrocarbon staple formed by two S5 via olefin metathesis). In acompetition fluorescence polarization assay, FP0025c displaced a labeledprobe from the axin site of β-catenin with an EC₅₀<100 nM. Anotherpeptide, FPO217c (Ac-AR₈ILDAHIM_(B)RVW (SEQ ID NO: 280), with N-terminalproline removed and valine replaced with isoleucine compared to FP0025c)was prepared. FPO217c Isomer 2 displayed >10-fold better potency thanFPO217c Isomer 1 in the competition FP assay; the reduced peptide(FPO217rc) was equivalent to Isomer 2.

Additionally or alternatively, binding to beta-catenin may be measuredby Surface Plasmon Resonance. In an exemplary assay, approximately 6nmol dried peptide diluted in buffer (50 mM Tris pH 8.0, 300 mM NaCl, 2%glycerol, 0.5 mM TCEP, 0.5 mM EDTA, 0.005% Tween-20, 1 mg/mL CM Dextran,0.02% w/v sodium azide) was assayed on a Biacore X100 using the BiacoreBiotin CAPture Kit (GE Healthcare) and biotinylated beta-catenin.Results were analyzed using the Biacore X100 Evaluation Software. Asmeasured, FP0025c displayed a Kd of 15 nM. FPO217c (Isomer 2) bound tothe armadillo domain of β-catenin with a K_(d) of 2 nM. FP0597c showed aKd of 7 nm. Additional exemplary data were presented in FIG. 1 and Table2.

Example 3. Provided Agents are Active in Cells

As appreciated by a person having ordinary skill in the art, varioustechnologies can be utilized to assess activities of provided agents,e.g., stapled peptides, in accordance with the present disclosure, e.g.,those described in the present disclosure, in WO 2017/062518, etc.

In some embodiments, a provided assay is a TCF/LEF reporter assay. Insome embodiments, in an exemplary such assay, TCF/LEF Luciferasereporter HEK293 cell lines (BPS Bioscience) were treated with dilutionseries of provided peptides for 18 hours. 300 ng/mL of Wnt3a (Peprotech)was added to the cells for the final 6 hours of incubation. Luciferaseactivity was measured using Bright-Glo Luciferase Assay (Promega)according to manufacturer's protocol. Exemplary data were presented inFIG. 2 and Table 2. FPO217c exhibited an IC₅₀ of 0.743 uM. Asdemonstrated, provided stapled peptides comprising various e.g.,sequences, lengths, modifications, amino acid residues, staples, etc.,were active. Applicant notes that for the TCF/LEF reporter assay,subsequent efforts to reproduce results observed for certain peptidesdescribed herein did not yield the same results, and in some tests, didnot show activities under the specific conditions of those tests (e.g.,amounts and/or batches of reagents). Additional assays are beingperformed to assess the reproducibility of observed properties and/oractivities of such peptides. Applicant also notes that teachings of thepresent disclosure are not restricted to a particular mechanism ofaction of described agents. For example, in some embodiments, one ormore agents may have relevant biological effects that are not specificto any interaction with (or lack of interaction with) beta-catenin orany particular site thereon.

Example 4. Provided Agents Modulate Gene Expression

As appreciated by those skilled in the art, beta-catenin regulatesexpression of many genes. Many conditions, disorders, and/or diseasesare associated with aberrant gene expression, including those connectedto one or more beta-catenin functions (e.g., regulated by beta-catenin).In some embodiments, as demonstrated by exemplary data herein, providedtechnologies can modulate expression of a variety of genes, includinginhibition of beta-catenin target genes in various cell lines includinga number of types of cancer cells.

Many technologies are known in the art, for example, qPCR, can be usedto assess levels and/or variations of gene expression and can beutilized in accordance with the present disclosure. In an exemplary qPCRassay, cells, e.g., HCT-116, DLD-1, were treated with a dilution seriesof provided peptides for a period of time, e.g., 18 hours. Total RNA wasextracted using, e.g., commercially available kit such as RNeasy Pluskit (Qiagen) according to manufacturer's protocols, and reversetranscribed to cDNA using, e.g., SuperScript Vilo IV master mix(ThermoFisher Scientific). Gene expression levels were determined byqPCR using, e.g., Taqman probes (Applied Biosciences) and TaqmanAdvanced Fast Master Mix (Applied Biosciences) on a QuantStudio 3(Applied Biosciences). Relative expression was quantified using delta Ctmethod. Exemplary data were presented in FIGS. 3, 4 and 5. In someembodiments, the following reagents were used for qPCR in the examples.In some embodiments, a control for normalization is beta-actin. Methodsfor qPCR, including design of primers and probes, are widely known andcan be utilized in accordance with the present disclosure.

Gene Assay ID Dye Label Scale Cat #* ACTB Hs01060665_g1 FAM-MGB 250rxn4331182 CTNNB1 Hs00355045_m1 FAM-MGB 250rxn 4331182 BIRC5 Hs04194392_s1FAM-MGB 250rxn 4331182 CCND1 Hs00765553_m1 FAM-MGB 250rxn 4331182 CD44Hs00153304_m1 FAM-MGB 250rxn 4331182 AXIN2 Hs00610344_m1 FAM-MGB 250rxn4331182 MYC Hs00153408_m1 FAM-MGB 250rxn 4331182 LEF1 Hs01547250_m1FAM-MGB 250rxn 4331182 LRP6 Hs00233945_m1 FAM-MGB 250rxn 4331182 VEGFAHs00900055_m1 FAM-MGB 250rxn 4331182 GAPDH Hs02786624_g1 TAM-MGB 250rxn4331182 Human GUSB Endogenous VIC/TAMRA 2500rxn  4310888E Control HumanB2M Endogenous Control VIC/TAMRA 2500rxn  4310886E *ThermoFisherScientific.

Example 5. Provided Agents have Improved Properties

Among other things, provided agents, e.g., stapled peptides, haveimproved properties, including solubility, pharmacokinetic properties,etc.

Among other things, the present disclosure recognizes that one of thechallenges associated with stapled peptides for use as therapeutics issolubility. In some embodiments, certain stapled peptides, e.g., thosecomprising hydrocarbon staples, have relatively low solubility. Asappreciated by those skilled in the art, low solubility can negativelyimpact, e.g., formulation, delivery, efficacy, etc. In some embodiments,the present disclosure provides technologies to improve solubility ofimproved stapled peptides. In some embodiments, the present disclosureprovides stapled peptides with solubility of at least 50, 60, 70, 80,90, 100, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,270, 280, 290, 300, 350, 400, 450, 500 uM in DPBS buffer (per liter, 8 gSodium Chloride, 0.2 g Potassium Phosphate, monobasic, 1.15 g SodiumPhosphate, dibasic, and 0.2 g Potassium Chloride).

Suitable assays for assessing solubility are widely known in the art andcan be utilized in accordance with the present disclosure. In someembodiments, in an exemplary protocol, dried peptide was reconstitutedin DPBS (DPBS, 1×, cell culture grade, Sigma D8537) in triplicate,vortexed, sonicated and then centrifuged. Absorbance of the supernatantwas measured at A280 (Nanodrop 2000) and the concentration wasdetermined using the extinction coefficient for tryptophan. Exemplarysolubility data are presented in, e.g., Table 2.

Example 6. Provided Agents have Improved Pharmacological Properties

Among other things, provided agents, e.g., stapled peptides, haveimproved properties, including solubility, pharmacokinetic properties,etc. In some embodiments, provided compounds demonstrate, among otherthings, improved half-life in animals.

Various technologies can be utilized to assess properties of providedagents, e.g., stapled peptides, in accordance with the presentdisclosure. In some embodiments, plasma PK methods are used to assesspharmacokinetic properties. In an exemplary assay, peptides wereformulated in 10% DMSO:90% saline and dosed by IV at 0.5 mg/kg percompound in three male Sprague-Dawley rats. Serial bleed time-pointswere taken at 2 min, 6 min, 10 min, 15 min, 30 min, 1, 2, 4, 6, 8, 12and 24 h and analyzed by quantitative LC/MS using a Thermo Q-ExactiveFocus LC/MS/MS. Samples were prepared by protein precipitation withMeOH. Data were fit to a two-compartment model. In one assay, FP0217c(Isomer 2) showed a plasma half-life of >1 hour, and FP0597c displayed ashorter plasma half-life. Exemplary data are presented, e.g., in FIG. 6.

Example 7. Provided Agents can Selectively Modulate Beta-CateninInteractions with Axin Over Other Entities

In some embodiments, the present disclosure provides agents, e.g.stapled peptides, that selectively bind to one or more beta-cateninsites that interact with Axin. In some embodiments, provided agents,e.g., stapled peptides, selectively compete with interactions with oneor more beta-catenin sites that interact with Axin. Particularly, insome embodiments, provided agents selectively modulate interactions withAxin at one or more beta-catenin sites that interact with Axin comparedto those at one or more beta-catenin sites that interact with BCL9. Insome embodiments, provided agents selectively disrupt beta-catenininteractions with proteins whose beta-catenin interacting sites areidentical or overlap with one or more sites that interact with Axin overthose whose beta-catenin interacting sites are identical or overlap withone or more sites that interact with BCL9. In some embodiments, providedagents selectively modulate beta-catenin interaction with Axin overbeta-catenin interaction with BCL9. In some embodiments, provided agentsselectively disrupts beta-catenin interaction with Axin overbeta-catenin interaction with BCL9. In some embodiments, a providedagent, e.g., a stapled peptide, has EC50 for disrupting interactionsbetween beta-catenin and BCL9 (or a probe, e.g.,Ac-Leu-Ser-Gln-Glu-Gln-Leu-Glu-His-Arg-Glu-Arg-Ser-Leu-Gln-Thr-Leu-Arg-Asp-Ile-Gln-Arg-nLeu-Leu-2NapA-bala-bala-Lys5FAM-NH2(SEQ ID NO: 2) (from Biochemistry, 2009, 48 (40), pp 9534-9541)) that isat least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000 or more fold of itsEC50 for disrupting interactions between beta-catenin and Axin (or aprobe, e.g., FITC-PEG1-PQ-S5-ILD-S5-HVRRVWR (SEQ ID NO: 1) (hydrocarbonstaple formed by two S5 via olefin metathesis) and/orFITC-bA-PQ-S5-ILD-S5-HVRRVWR (SEQ ID NO: 3) (hydrocarbon staple formedby two S5 via olefin metathesis)) as measured by, e.g., a competitionfluorescence polarization assay. In some embodiments, provided agents,e.g., stapled peptides, do not observably disrupt interactions betweenbeta-catenin and BCL9. In such cases, EC50 may not be determinable, butas appreciated by those skilled in the art, can be treated as at least2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 1000 or more fold of a determinableEC50 from a detectable disruption.

Various technologies can be utilized to assess interactions withbeta-catenin at sites, e.g., that interact with BCL9 or Axin. In someembodiments, competition fluorescence polarization is utilized to assessinteraction/modulation selectivity. In some embodiments, a competitionfluorescence polarization assay for Axin sites (e.g., beta-catenin sitesthat interacts with Axin) was as described above. In some embodiments,e.g., for assess selectivity between BCL9 sites and Axin sites, acompetition fluorescence polarization assay for Axin and/or BCL9 sitesmay be employed. In some embodiments, in an exemplary BCL9 competitionFP assay, peptide solutions were prepared in a buffer (e.g., 50 mM TrispH 8.0, 250 mM NaCl, 2% glycerol, 0.5 mM EDTA, 0.02% w/v sodium azide)using a, e.g., 3-fold, serial dilution from, e.g., 10 μM. Probe solution(e.g., 250 nM full-length 8-Catenin, 20 nM 5FAM labeled peptide inbuffer) was prepared and a volume of, e.g., 40 μL, per well plated in asuitable multi-well plate, e.g., a black polystyrene 384-well plate(Corning). A suitable volume, e.g., an equal volume of the serialdiluted peptide was added to the plate and incubated protected fromlight for a period of time, e.g., 15 minutes prior to read. Reads wereperformed, e.g., on a Spectramax M5 (Molecular Devices) in duplicate. Insome embodiments, a probe isAc-Leu-Ser-Gln-Glu-Gln-Leu-Glu-His-Arg-Glu-Arg-Ser-Leu-Gln-Thr-Leu-Arg-Asp-Ile-Gln-Arg-nLeu-Leu-2NapA-bala-bala-Lys5FAM-NH2(SEQ ID NO: 2) (from Biochemistry, 2009, 48 (40), pp 9534-9541). Asdemonstrated, e.g., by exemplary data in FIG. 7, in some embodiments,provided stapled peptides selectively disrupts interactions at one ormore Axin sites over those at one or more BCL9 sites.

Example 8. Preparation of Stapled Peptides with Diverse StructuralElements and Assessment of their Properties

Among other things, the present disclosure provides various structuralelements, including of those of the staples, such as chemistry(hydrocarbon linker v. non-hydrocarbon linker), positioning (positionsof staple connection, (i, i+4), (i, i+7), etc.), lengths,stereochemistry, etc., and combinations thereof, that can be utilized todesign and prepare stapled peptides with significantly improvedproperties and/or activities. Various structural elements can alsoimpact preparation of stapled peptides in terms of yield, purity,selectivity, etc. The present example illustrates preparation of stapledpeptides with diverse structures using different reaction conditions.Among other things, certain structural features, e.g., those of staples(types, lengths, etc.), that can provide various advantages (preparationyield, purity, selectivity, binding affinity, etc.) are identified. Insome embodiments, exemplary stapled peptides has better properties,e.g., solubility, binding affinity, cell permeability, etc. than StAxstapled peptides reported in reported in Grossmann et al. PNAS 10917942-17947. In some embodiments, exemplified stapled peptides containamino acid sequences that are highly homologous to StAx33 of Grossman.

A number of stapled peptides were prepared, with staple length of about10-14 for carbamate staples and 8-12 for amino staples (which in thiscase comprising —N(R)— not bonded to —C(O)—). In some embodiments, somestapled peptides are double stapled (in some cases, stiched peptided).Table 4A illustrates certain results using 2× 30 mol % Grubbs I, at 40°C., 2 hrs. Table 4B illustrates certain results using 2× 5 mol %Hoveyda-Grubbs II, at 60° C., 2 hrs. Az is monomer A. PyrS is Monomer B.PyrR is Monomer C. SgN is Monomer D. RgN is Monomer E. SdN is Monomer F.RdN is Monomer G. SeN is Monomer H. ReN is Monomer I.

In some embodiments, staple length of 11 or more may deliver higheryields compared to a shorter staple length (e.g., forazetidine-containing stapled peptides as illustrated). In someembodiments, a preferred staple length is 11 or more. In someembodiments, staple length of 10-14 or more may deliver higher yieldscompared to a staple of another length (e.g., for pyrrolidine-containingstapled peptides as illustrated). In some embodiments, a preferredstaple length is 11 or more. In some embodiments, a preferred staplelength is 10-14. In some embodiments, pyrrolidine-containing staplesgenerally are slower to form compared to azetidine-containing staplesunder comparable conditions. In some embodiments, for acyclic amines,amino acid residues comprising olefin in a hydrocarbon side chain at theN-terminus position typically resulted in lower olefin metathesisproduct formation.

FP EC50 data of certain stapled peptides were presented in Table 5.

For amino staple formation reaction, most reactions yielded clean aminostapled peptides under the condition used, with a few exceptions wheremultiple products and/or double isomers were observed. Exemplary resultswere presented in Table 6. In some embodiments, acyclic amino stapleswere more difficult to form compared to cyclic amino staples undercertain conditions.

Exemplary FP EC₅₀ data were presented in Table 5. In some embodiments,stapled peptides with amino staples have lower binding affinity thanstapled peptides with other types of staples, e.g., carbamate staples(in one case, FP-0738c (1800 nM) vs. FP-0738a (200 nM)).

In some embodiments, the following staples provided better results andmay be preferred (exemplary stapled peptides in parentheses):

Carbamate Staples:

Az/R6 (FP-0725c)

PR/R6 (FP-0745c)

S7/PS (FP-0763c)

R4/PR (FP-0765c)

R5/PR (FP-0766c)

R6/PR (FP-0767c)

R7/PR (FP-0768c)

Staple Length=11 to 14 atoms

Amino Staples:

S7/Az (FP-0738a)

PR/R6 (FP-0745a)

Staple Length=11 to 12 atoms

By a Surface Plasmon Resonance—Biacore assay, R4/PR (FP-0765c) displayeda Kd about 13 nM, S5/S5 (FP-0787c) Kd about 14 nM, R5/PR (FP-0766c) Kdabout 7 nM, Az/R6 (FP-0725c) Kd about 22 nM, S7/Az (FP-0738a) Kd about43 nM, and PR/R₆ (FP-0745a) Kd about 34 nM.

Example 9. Additional Methods for Olefin Metathesis

In some embodiments, the present disclosure provides methods forpreparing stapled peptides. In some embodiments, the present disclosureprovides methods for preparing stapled peptides, comprising forming astaple through olefin metathesis. In some embodiments, the presentdisclosure provides methods for ring closing metathesis to form astaple.

Various metathesis catalysts may be utilized in accordance with thepresent disclosure. In some embodiments, a catalyst is a Ru-catalyst. Insome embodiments, a Ru-catalyst is Grubbs I, Grubbs II, Hoveyda-Grubbs Iand Hoveyda-Grubbs II. In some embodiments, catalyst loading is 5 mol %.In some embodiments, catalyst loading is 20 mol %. In some embodiments,Hoveyda-Grubbs II provides better results than one or more othercatalysts.

In some embodiments, 11 substrate peptides for olefin metathesis wereused to evaluate various conditions, each of which can be fully stapledwith a single 30 mol % Grubbs I treatment. Exemplary results werepresented in FIG. 8.

For Grubbs I, one treatment in DCE, at 40° C., 2 hrs, all reactionsusing 20 mol % were complete with 7 yielding no or traces of byproductwhile 4 produced 17% to 50% byproduct. Single treatment with 5 mol % wasnot sufficient to completely staple peptides, with 3 peptides alsoshowing byproduct formation. For Grubbs II, one treatment in DCE, at 40°C., 2 hrs, lower efficiency was observed compared to Grubbs I, nocomplete reaction was observed, by-product formation was observed, andstarting material was the major species throughout. For Hoveyda-GrubbsI, one treatment in DCE, at 40° C., 2 hrs, no complete reactions wereobserved, and either trace or no stapled product observed with 5 mol %.For Hoveyda-Grubbs II, one treatment in DCE, at 60° C., 2 hrs, allpeptides were fully stapled with 5 mol % of Hoveyda-Grubbs II, and lowerbyproduct to product ratio than with other catalysts.

In some embodiments, an optimized process is

In some embodiments, a pre-optimized process is

Exemplary results were presented below.

SEQ 1st Treatment ID (5 mol % HGII ID NO: Peptide Sequence at 60 C)Isomers FP-0996 281 Ac-HRERSLQTLR-Az-IQR-R6-LF-NH2 Rxn complete; traceof Single unstapled FP-0997 282 Ac-HRERSLQTLR-S5-IQR-Az-LF-NH2 50%stapled Single FP-0998 283 Ac-HRERSLQTLR-P_(R)-IQR-R5-LF-NH2 15%unstapled Single remaining FP-0999 284 Ac-HRERSLQTLR-R6-IQR-P_(R)-LF-NH230% unstapled Single remaining FP-1000 285Ac-HRE-Az-SLQ-R6-LRDIQR-Nle-LF- Rxn complete; 13% Single NH2 byproductFP-1001 286 Ac-HRE-S5-SLQ-Az-LRDIQR-Nle-LF- 40% stapled Single NH2FP-1002 287 Ac-HRE-P_(R)-SLQ-R5-LRDIQR-Nle-LF- 10% unstapled Single NH2remaining FP-1003 288 Ac-HRE-R6-SLQ-P_(R)-LRDIQR-Nle-LF- 13% unstapledSingle NH2 remaining FP-1004 289 Ac-Az-HRE-R6-SLQ-R8-LRDIQR-P_(S)- Rxncomplete; trace of Double LF-NH2 unstapled (2:1) FP-1005 290Ac-S5-HRE-Az-SLQ-R8-LRDIQR-P_(S)- 50% stapled Double LF-NH2 (2:1)FP-1006 291 Ac-P_(R)-HRE-R5-SLQ-R8-LRDIQR-P_(S)- 10% unstapled DoubleLF-NH2 remaining (2:1) FP-1007 292 Ac-R6-HRE-P_(R)-SLQ-R8-LRDIQR-P_(S)-15% unstapled Double LF-NH2 remaining (2:1) SEQ 1st Treatment 2ndTreatment ID (5 mol % HGII at (5 mol % HGII ID NO: Peptide Sequence 60C) at 60 C) FP-0719 293 Ac-Pro-Gln-M_(A)-Ile-Leu-Asp-R3-His- traces ofstapled NA Val-Arg-Arg-Val-Trp-Arg-NH2 FP-0720 294Ac-Pro-Gln-M_(A)-Ile-Leu-Asp-S3-His- no reaction NAVal-Arg-Arg-Val-Trp-Arg-NH2 FP-0726 295Ac-Pro-Gln-M_(A)-Ile-Leu-Asp-S6-His- 10% stapled NAVal-Arg-Arg-Val-Trp-Arg-NH2 product; double isomers FP-0741 296Ac-Pro-Gln-M_(C)-Ile-Leu-Asp-R4-His- traces of stapled NAVal-Arg-Arg-Val-Trp-Arg-NH2 FP-0748 297Ac-Pro-Gln-M_(C)-Ile-Leu-Asp-S7-His- traces of stapled NAVal-Arg-Arg-Val-Trp-Arg-NH2 FP-0761 298Ac-Pro-Gln-S6-Ile-Leu-Asp-M_(B)-His- 60% stapled rxn complete;Val-Arg-Arg-Val-Trp-Arg-NH2 traces of unstapled FP-0763 299Ac-Pro-Gln-S7-Ile-Leu-Asp-M_(B)-His- 42% stapled rxn complete;Val-Arg-Arg-Val-Trp-Arg-NH2 traces of unstapled FP-0769 300Ac-Pro-Gln-S5-Ile-Leu-Asp-M_(D)-His- 20% stapled 46% stapled;Val-Arg-Arg-Val-Trp-Arg-NH2 double isomers FP-0770 301Ac-Pro-Gln-S5-Ile-Leu-Asp-M_(F)-His- 50% stapled; double NAVal-Arg-Arg-Val-Trp-Arg-NH2 isomers FP-0771 302Ac-Pro-Gln-S5-Ile-Leu-Asp-M_(H)-His- 25% stapled; double NAVal-Arg-Arg-Val-Trp-Arg-NH2 isomers FP-0772 303Ac-Pro-Gln-R5-Ile-Leu-Asp-M_(D)-His- 20% stapled 50% stapledVal-Arg-Arg-Val-Trp-Arg-NH2 FP-0773 304Ac-Pro-Gln-R5-Ile-Leu-Asp-M_(F)-His- no reaction NAVal-Arg-Arg-Val-Trp-Arg-NH2 FP-0774 305Ac-Pro-Gln-R5-Ile-Leu-Asp-M_(H)-His- no reaction NAVal-Arg-Arg-Val-Trp-Arg-NH2 FP-0775 306Ac-Pro-Gln-R5-Ile-Leu-Asp-M_(E)-His- Val-Arg-Arg-Val-Trp-Arg-NH2 FP-0776307 Ac-Pro-Gln-R5-Ile-Leu-Asp-M_(G)-His- 60% stapled rxn complete;Val-Arg-Arg-Val-Trp-Arg-NH2 traces of unstapled FP-0777 308Ac-Pro-Gln-R5-Ile-Leu-Asp-M_(I)-His- 60% stapled rxn complete;Val-Arg-Arg-Val-Trp-Arg-NH2 traces of unstapled FP-0778 309Ac-Pro-Gln-M_(D)-Ile-Leu-Asp-S5-His- 58% stapled rxn complete;Val-Arg-Arg-Val-Trp-Arg-NH2 traces of unstapled FP-0779 310Ac-Pro-Gln-M_(F)-Ile-Leu-Asp-S5-His- 42% stapled rxn complete;Val-Arg-Arg-Val-Trp-Arg-NH2 traces of unstapled FP-0780 311Ac-Pro-Gln-M_(H)-Ile-Leu-Asp-S5-His- ~33% stapled, rxn complete;Val-Arg-Arg-Val-Trp-Arg-NH2 coelutes with −M_(H) traces of truncationunstapled FP-0781 312 Ac-Pro-Gln-M_(E)-Ile-Leu-Asp-R5-His-Val-Arg-Arg-Val-Trp-Arg-NH2 FP-0782 313Ac-Pro-Gln-M_(G)-Ile-Leu-Asp-R5-His- 33% stapled rxn complete;Val-Arg-Arg-Val-Trp-Arg-NH2 traces of unstapled FP-0783 314Ac-Pro-Gln-M_(I)-Ile-Leu-Asp-R5-His- 56% stapled rxn complete;Val-Arg-Arg-Val-Trp-Arg-NH2 traces of unstapled FP-0784 315Ac-Pro-Gln-M_(E)-Ile-Leu-Asp-S5-His- Val-Arg-Arg-Val-Trp-Arg-NH2 FP-0785316 Ac-Pro-Gln-M_(G)-Ile-Leu-Asp-S5-His- traces of stapled NAVal-Arg-Arg-Val-Trp-Arg-NH2 FP-0786 317Ac-Pro-Gln-M_(I)-Ile-Leu-Asp-S5-His- traces of stapled NAVal-Arg-Arg-Val-Trp-Arg-NH2 NA: data not available/not performed.

In some embodiments, a “stiched” stapled peptides is selected frombelow:

Ac-PyrS-HRE-B5-SLQ-PyrR-LRDIQR- Ac-HRERSL-PyrS-TLR-B5-IQR-PyrR-LF-Nle-LF-NH2 (SEQ ID NO: 318) NH2 (SEQ ID NO: 325)Ac-SgN-HRE-B5-SLQ-RdN-LRDIQR-Nle- Ac-HRERSL-SgN-TLR-B5-IQR-RdN-LF-LF-NH2 (SEQ ID NO: 319) NH2 (SEQ ID NO: 326)Ac-SdN-HRE-B5-SLQ-RdN-LRDIQR-Nle- Ac-HRERSL-SdN-TLR-B5-IQR-RdN-LF-LF-NH2 (SEQ ID NO: 320) NH2 (SEQ ID NO: 327)Ac-SeN-HRE-B5-SLQ-RdN-LRDIQR-Nle- Ac-HRERSL-SeN-TLR-B5-IQR-RdN-LF-LF-NH2 (SEQ ID NO: 321) NH2 (SEQ ID NO: 328)Ac-SgN-HRE-B5-SLQ-ReN-LRDIQR-Nle- Ac-HRERSL-SgN-TLR-B5-IQR-ReN-LF-LF-NH2 (SEQ ID NO: 322) NH2 (SEQ ID NO: 329)Ac-SdN-HRE-B5-SLQ-ReN-LRDIQR-Nle- Ac-HRERSL-SdN-TLR-B5-IQR-ReN-LF-LF-NH2 (SEQ ID NO: 323) NH2 (SEQ ID NO: 330)Ac-SeN-HRE-B5-SLQ-ReN-LRDIQR-Nle- Ac-HRERSL-SeN-TLR-B5-IQR-ReN-LF-LF-NH2 (SEQ ID NO: 324) NH2 (SEQ ID NO: 331)

As described in the present disclosure, provided agents, e.g., stapledpeptides, have a number of significantly improved properties andactivities, in some embodiments particularly when compared to one ormore appropriate reference agents. Among other things, improvedstability, increased solubility, increased cell permeability, increaseactivities, increased selectivity, and/or lowered toxicities, wereobserved when compared to a number of reference agents, e.g., unstapledpeptides, small molecule Wnt pathway inhibitors, stapled peptidescomprising hydrocarbon staples, stapled peptides not interacting withone or more beta-catenin sites that interact with Axin (e.g., stapledpeptides interacting with one or more beta-catenin sites that interactwith BCL9 but not Axin). A number of assays, including those describedin the present disclosure and variations thereof, can be utilized toassess one or more properties and activities of provided agents, e.g.,stapled peptides.

TABLE 2 Exemplary data. in vitro TCF/LEF Reporter FP EC₅₀ SolubilityK_(d) by SPR IC₅₀ Inhibition at Peptide (nM)* (mM) (nM) (mM) 10 μMFP0001c FP0003c FP0005c +++ 45% FP0006a +++ 10% FP0007c 54% FP0009c 54%FP0011c 34% FP0025c +++ 15 65% FP0098 18% FP0099 38% FP0110 19% FP0212sIsomer 2 ++ 35% FP0216c 37% FP0217a NB 111  0% FP0217c Isomer 1 +FP0217c Isomer 2 +++ 7 2 0.743 72% c14-FP0217a c14-FP0217c 54%c16-FP0217a FP0217c_bAfree +++ 155 9 FP0217c_btn +++ FP0217c_cl8aFP0217rc +++ 26 FP0217s Isomer 1 +++ 5 FP0217s Isomer 2 +++ 2 FP0217u+++ FP0218c ++ 41% FP0219c ++ 20% FP0220c +++ 14 39% FP0221c 38% FP0222c22% FP0223a 18% FP0224a 15% FP0243c 35% FP0244c 68% FP0247c  0% FP0249c 0% FP0250c  0% FP0253c 63% FP0264c 59% FP0265c 64% FP0268c  0% FP0269c 0% FP0270c  0% FP0271c  0% FP0272c FP0273c  4% FP0274c 42% FP0278c  0%FP0279c 34% FP0280c 78% FP0281c 38% FP0282c 42% FP0284c  0% FP0285c 19%FP0286c  0% FP0290c 60% FP0292c 23% FP0293c 32% FP0295c 36% FP0296c 64%FP0298c 38% FP0299c  2% FP0300c 39% FP0302c 51% FP0306c 48% FP0317a 34%FP0318a FP0318c 48% FP0321c 59% FP0324c 51% FP0325a FP0325c 73% FP0327c 0% FP0335a ++  0% FP0335c Isomer 1 ++ 22% FP0335c Isomer 2 +++ 37%FP0336c 43% FP0338c ++ 3 36% FP0344c  0% FP0345c 43% FP0346c 36% FP0349c50% FP0350c  1% FP0352c 38% FP0353c  0% FP0354c 28% FP0355c  0% FP0357c37% FP0365c ++ FP0365c Isomer 1 + FP0365c Isomer 2 + FP0368c  0% FP0369c38% FP0371c 46% FP0380c 25% FP0383c 22% FP0391c FP0395c 32% FP0405c +FP0406c ++ FP0407c + FP0408c NB FP0409a FP0409c NB FP0409c_freec16-FP0409a c16-FP0409c FP0410c ++ FP0411c FP0412c FP0495a + 18% FP0495c++ 20% FP0501c ++ 33% FP0502a + 49% FP0502c Isomer 1 +++ 20% FP0502cIsomer 2 ++ 27% FP0503a ++ 236 32% FP0503c ++ 35 10% FP0506a + FP0506cIsomer 1 ++ FP0506c Isomer 2 +++ FP0507a +++ FP0507c + FP0509a + 192FP0509c +++ 32 3 FP0510a + 165 FP0510c Isomer 1 +++ 65 FP0510c Isomer 2+++ 31 FP0511a +++ 170 FP0511c Isomer 1 +++ 49 FP0511c Isomer 2 +++ 24FP0516a Isomer 1 ++ FP0516a Isomer 2 + FP0516c ++ FP0536c NB FP0537c NBFP0538c + FP0539c FP0539c Isomer 1 52% FP0539c Isomer 2 36% FP0540c NBFP0541c NB FP0542c + 18% FP0554c Isomer 1 NB 29% FP0554c Isomer 2 NB 35%FP0555c Isomer 1 NB 43% FP0555c Isomer 2 NB 32% FP0556c Isomer 1 NB 33%FP0556c Isomer 2 NB 38% FP0557c Isomer 1 NB 43% FP0557c Isomer 2 NB 30%FP0558c Isomer 1 NB 38% FP0558c Isomer 2 NB 40% FP0559c Isomer 1 NB 44%FP0559c Isomer 2 NB 31% FP0560c Isomer 1 NB 40% FP0560c Isomer 2 NB 22%FP0561c Isomer 1 NB 38% FP0561c Isomer 2 NB 35% FP0562c Isomer 1 NB 22%FP0562c Isomer 2 NB 32% FP0563c Isomer 1 NB 34% FP0563c Isomer 2 NB 30%FP0564c Isomer 1 NB 46% FP0564c Isomer 2 NB FP0565c Isomer 1 NB FP0565cIsomer 2 NB FP0566c Isomer 1 NB FP0567c Isomer 1 NB FP0567c Isomer 2 NBFP0568c Isomer 1 NB FP0568c Isomer 2 NB FP0569c Isomer 1 NB FP0569cIsomer 2 NB FP0570c Isomer 1 NB FP0570c Isomer 2 NB FP0571c Isomer 1 NBFP0571c Isomer 2 NB FP0572c Isomer 1 NB FP0573c Isomer 1 NB FP0573cIsomer 2 NB FP0574c Isomer 1 NB FP0574c Isomer 2 NB FP0575c Isomer 1 NBFP0575c Isomer 2 NB FP0576c Isomer 1 NB FP0576c Isomer 2 NB FP0577cIsomer 1 NB FP0578c Isomer 1 NB FP0578c Isomer 2 NB FP0587c +++ 33%FP0588c +++ 133 61% FP0594c ++ 166 23% FP0596c + 21% FP0597c +++ 98 41.021 81% FP0597c_c12 4 FP0597c_c8 2 FP0598c +++ 23% FP0601c +++ 30%FP0604c +++ 32% FP0605c +++ 30% FP0611c +++ 56% FP0616c +++ 56 51%FP0617c +++ 63 62% FP0625c ++ 20% FP0626c +++ 40 53% FP0628_aib ++ 88FP0629c + 49% FP0630c ++ 53% FP0631c +  0% FP0632c ++ 13 57% FP0633c +16% FP0634c +++ 45% FP0635c ++ 19% FP0636c ++ 41% FP0639c +++ 40%FP0640c +++ 25% FP0644c ++ 34% FP0645c +++ 26% FP0721a FP0721c + FP0723aFP0723c + FP0724c + FP0725a + FP0725c +++ 22 FP0727c + FP0728c +FP0731c + FP0733c + FP0734a NB FP0734c NB FP0735a + FP0735c + FP0736a ++FP0736c + FP0738a ++ 43 FP0738c + FP0743a NB FP0743c + FP0745a ++ 34FP0745c ++ FP0751a NB FP0751c + FP0752c + FP0753a NB FP0758a NBFP0758c + FP0761c ++ FP0763a + FP0763c ++ FP0765c +++ 13 N/A N/A FP0766c+++ 7 N/A N/A FP0767a + FP0767c ++ FP0768a NB FP0768c ++ FP0776c +FP0776a + FP0777c + FP0777a + FP0778c + FP0779c + FP0780c + FP0782c ++FP0783c + FP0783a + FP0787s ++ 14 *+++: < =100 nM EC50; ++: 100-500 nMEC50; +: 500-5000 nM EC50; N/A, N.D.: relevant values not determinedfrom currently available data collected from utilized assay conditions,e.g., dose ranges, concentrations, etc.; NB: no binding detected underutilized assay conditions.

TABLE 3 Exemplary results. Part A Metathesis Target Binding KdSolubility Beta-Catenin Luciferase Reporter ID Efficiency Binding (nM)in DPBS (μM) % Inhibition at 10 μM FP0512c fair N.D. N.D. N.D. N.D.FP0513c good N.D. N.D. N.D. N.D. FP0514c poor N.D. N.D. N.D. N.D.FP0515c fair N.D. N.D. N.D. N.D. FP0516c fair 151 N.D. N.D. 27% FP0517cpoor N.D. N.D. N.D. N.D. FP0335c fair Yes (Isomer 2 N.D. N.D. 22%(Isomer 1) more tightly than 37% (Isomer 2) Isomer 1) FP0492c fair N.D.N.D. N.D. N.D. FP0491c poor N.D. N.D. N.D. N.D. FP0490c poor N.D. N.D.N.D. N.D. FP0338c good Yes 10 N.D. 30% FP0495c good Yes N.D. N.D. 20%FP0494c poor N.D. N.D. N.D. N.D. FP0493c poor N.D. N.D. N.D. N.D.FP0499c fair N.D. N.D. N.D. N.D. FP0498c poor N.D. N.D. N.D. N.D.FP0497c poor N.D. N.D. N.D. N.D. FP0496c fair N.D. N.D. N.D. N.D.FP0503c fair Yes N.D.  35 10% FP0502c fair Yes N.D. N.D. 20% (Peak 1)27% (Peak 2) FP0501c fair Yes N.D. N.D. 33% FP0500c poor N.D. N.D. N.D.N.D. FP0507c good but two Yes N.D. N.D. 32% (Only one isomer isolated)isomers FP0506c fair Yes N.D. N.D. 36% FP0505c poor N.D. N.D. N.D. N.D.FP0504c poor N.D. N.D. N.D. N.D. FP0486c poor N.D. N.D. N.D. N.D.FP0485c poor N.D. N.D. N.D. N.D. FP0484c poor N.D. N.D. N.D. N.D.FP0483c poor N.D. N.D. N.D. N.D. FP0217c fair Yes (Isomer 2 4 (Isomer 2)12 (Isomer 2) 62% (Isomer 2) more tightly than Isomer 1) FP0489c fairN.D. N.D. N.D. N.D. FP0488c No data N.D. N.D. N.D. N.D. FP0487c poorN.D. N.D. N.D. N.D. FP0508c poor N.D. N.D. N.D. N.D. FP0509c fair Yes  3 42 39% FP0510c good but two Yes (Isomer 2 N.D. 65 (Isomer 1) 22%(Isomer 1) isomers more tightly than 31 (Isomer 2) 33% (Isomer 2)Isomer 1) FP0511c good but two Yes (Isomer 2 N.D. 49 (Isomer 1) 17%(Isomer 1) isomers more tightly than 24 (Isomer 2) 18% (Isomer 2)Isomer 1) FP0520c good N.D. N.D. N.D. N.D. FP0521c fair N.D. N.D. N.D.N.D. FP0522c good but two N.D. N.D. N.D. N.D. isomers FP0523c good N.D.N.D. N.D. N.D. FP0617c good Yes N.D.  63 62% FP0616c good Yes N.D.  5651% FP0615c poor N.D. N.D. N.D. N.D. FP0611c good Yes N.D. N.D. 58%FP0623c poor N.D. N.D. N.D. N.D. FP0624c poor N.D. N.D. N.D. N.D.FP0625c fair Yes N.D. N.D.  0% FP0592c good but 2 N.D. N.D. N.D. N.D.isomers FP0599c good N.D. N.D. N.D. N.D. FP0606c poor N.D. N.D. N.D.N.D. FP0627c fair N.D. N.D. N.D. N.D. FP0618c poor N.D. N.D. N.D. N.D.FP0619c poor N.D. N.D. N.D. N.D. FP0613c poor N.D. N.D. N.D. N.D.FP0609c poor N.D. N.D. N.D. N.D. FP0610c poor N.D. N.D. N.D. N.D.FP0612c poor N.D. N.D. N.D. N.D. FP0614c poor N.D. N.D. N.D. N.D.FP0620c poor N.D. N.D. N.D. N.D. FP0621c poor N.D. N.D. N.D. N.D.FP0622c fair N.D. N.D. N.D. N.D. FP0587c good Yes N.D. N.D. 43% FP0588cgood but 2 Yes N.D. 133 61% isomers FP0589c good but 2 N.D. N.D. N.D.N.D. isomers FP0590c good but 2 N.D. N.D. N.D. N.D. isomers FP0591c goodbut 2 N.D. N.D. N.D. N.D. isomers FP0593c good but 2 N.D. N.D. N.D. N.D.isomers FP0594c good Yes N.D. 166 23% FP0595c fair N.D. N.D. N.D. N.D.FP0596c good Yes N.D. N.D. 11% FP0597c good Yes  4  98 81% FP0598c goodYes N.D.  91 23% FP0600c good but 2 N.D. N.D. N.D. N.D. isomers FP0601cgood Yes N.D. N.D. 40% FP0602c fair N.D. N.D. N.D. N.D. FP0603c poorN.D. N.D. N.D. N.D. FP0604c good Yes N.D. N.D. 46% FP0605c good Yes N.D.N.D. 50% FP0607c poor N.D. N.D. N.D. N.D. FP0608c fair N.D. N.D. N.D.N.D. FP0626c good Yes N.D.  40 60% Part B Carbamate- Amino- Beta-CateninLuciferase Stapled Starting Stapled Solubility in DPBS Reporter %Inhibition at Material Product CO₂ Extrusion Target Binding (μM) 10 μMFP0516c FP0516a Yes (Two isomers Yes (Isomer 1 binds N.D. N.D. isolated)more tightly than Isomer 2) FP0335c FP0335a Yes Yes N.D. 0% FP0338cFP0338a Yes N.D. N.D. N.D. FP0495c FP0495a Yes Yes N.D. 18% FP0503cFP0503a Yes Yes 236 32% FP0502c FP0502a Yes No N.D. 49% FP0507c FP0507aYes Yes N.D. 32% FP0506c FP0506a Yes No N.D.  0% FP0217c FP0217a Yes No111  0% FP0509c FP0509a Yes Yes 192  0% FP0510c FP0510a Yes Yes 165 49%FP0511c FP0511a Yes Yes 170  0% Poor: <1:2 stapled:unstapled or <1:1stapled:unstapled with two isomers Fair: between approx. 1:2stapled:unstapled and 2:1 stapled:unstapled, potentially with twoisomers (also in this category is combinations that gave up to 3:1 or sostapled:unstapled but gave either two major isomers or significantamount of neither stapled nor unstapled byproduct) Good: better than 2:1stapled:unstapled with one major isomer Good but two isomers: Betterthan approx. 4:1 stapled but with two major isomers N.D.—Not determinedor not presented in this Table.

TABLE 4 Exemplary results (Table 4 discloses SEQ ID NOS 293, 294, 229,231, 232, 234, 295, 235, 236, 332, 333, 237, 238, 240, 242, 244, 334,246, 335, 296, 248, 336, 250, 337, 297, 338, 252, 253, 339, 340, 256,341, 342, 257, 259, 343, 260, 261, 263, 265, 300-305, 266, 268, 270-274,316 and 317, respectively, in order of appearance). Com- In- plete Com-com- with plete plete By- single after 2 or No product Peptide treat-treat- Re- forma- Double ID Sequence ment ments action tion Isomer A-2x30 mol % Grubbs I, at 40° C., 2 hrs Azetidine Carbamate Staples FP-0719FP-0720 FP-0721 FP-0723 FP-0724 FP-0725 FP-0726 FP-0727Ac-P-Q-Az-I-L-D-R3-H-V-R-R-V-W-R-NH2Ac-P-Q-Az-I-L-D-S3-H-V-R-R-V-W-R-NH2Ac-P-Q-Az-I-L-D-R4-H-V-R-R-V-W-R-NH2Ac-P-Q-Az-I-L-D-R5-H-V-R-R-V-W-R-NH2Ac-P-Q-Az-I-L-D-S5-H-V-R-R-V-W-R-NH2Ac-P-Q-Az-I-L-D-R6-H-V-R-R-V-W-R-NH2Ac-P-Q-Az-I-L-D-S6-H-V-R-R-V-W-R-NH2Ac-P-Q-Az-I-L-D-R7-H-V-R-R-V-W-R-NH2       x   x   x     x   x x x        x       25%       50%             x

FP-0728 FP-0729 FP-0730 FP-0731 FP-0733 FP-0734 FP-0735 FP-0736 FP-0737FP-0738 Ac-P-Q-Az-I-L-D-S7-H-V-R-R-V-W-R-NH2Ac-P-Q-R3-I-L-D-Az-H-V-R-R-V-W-R-NH2Ac-P-Q-S3-I-L-D-Az-H-V-R-R-V-W-R-NH2Ac-P-Q-R4-I-L-D-Az-H-V-R-R-V-W-R-NH2Ac-P-Q-R5-I-L-D-Az-H-V-R-R-V-W-R-NH2Ac-P-Q-S5-I-L-D-Az-H-V-R-R-V-W-R-NH2Ac-P-Q-R6-I-L-D-Az-H-V-R-R-V-W-R-NH2Ac-P-Q-S6-I-L-D-Az-H-V-R-R-V-W-R-NH2 Ac-P-Q-R7-IL-D-Az-H-V-R-R-V-W-R-NH2 Ac-P-Q-S7-I-L-D-Az-H-V-R-R-V-W-R-NH2          x x x   x x       x       x   x x x (80%) 28%               22%

Pyrrolidine Carbamate Staples FP-0739 FP-0741 FP-0743 PP-0744 FP-0745FP-0746 FP-0748 FP-0749 FP-0751 FP-0752Ac-P-Q-P_(R)-I-L-D-R3-H-V-R-R-V-W-R-NH2Ac-P-Q-P_(R)-I-L-D-R4-H-V-R-R-V-W-R-NH2Ac-P-Q-P_(R)-I-L-D-R5-H-V-R-R-V-W-R-NH2Ac-P-Q-P_(R)-I-L-D-S5-H-V-R-R-V-W-R-NH2Ac-P-Q-P_(R)-I-L-D-R6-H-V-R-R-V-W-R-NH2Ac-P-Q-P_(R)-I-L-D-S6-H-V-R-R-V-W-R-NH2Ac-P-Q-P_(R)-I-L-D-S7-H-V-R-R-V-W-R-NH2Ac-P-Q-P_(S)-I-L-D-S3-H-V-R-R-V-W-R-NH2Ac-P-Q-P_(S)-I-L-D-S5-H-V-R-R-V-W-R-NH2Ac-P-Q-P_(S)-I-L-D-S6-H-V-R-R-V-W-R-NH2     x   x                 x x xx   x   x x x

FP-0753 Ac-P-Q-P_(S)-I-L-D-S7-H-V-R-R-V-W-R-NH2 x 28% FP-0756 FP-0758FP-0759 FP-0760 FP-0761 FP-0763 FP-0764 FP-0765 FP-0766Ac-P-Q-R4-I-L-D-P_(S)-H-V-R-R-V-W-R-NH2Ac-P-Q-R5-I-L-D-P_(R)-H-V-R-R-V-W-R-NH2Ac-P-Q-S5-I-L-D-P_(R)-H-V-R-R-V-W-R-NH2Ac-P-Q-R6-I-L-D-P_(S)-H-V-R-R-V-W-R-NH2Ac-P-Q-S6-I-L-D-P_(S)-H-V-R-R-V-W-R-NH2Ac-P-Q-S7-I-L-D-P_(R)-H-V-R-R-V-W-R-NH2Ac-P-Q-R3-I-L-D-P_(R)-H-V-R-R-V-W-R-NH2Ac-P-Q-R4-I-L-D-P_(R)-H-V-R-R-V-W-R-NH2Ac-P-Q-R5-I-L-D-P_(R)-H-V-R-R-V-W-R-NH2     x           x         x x  x x x (80%)   x     x     x

FP-0767 Ac-P-Q-R6-I-L-D-P_(R)-H-V-R-R-V-W-R-NH2 x FP-0768Ac-P-Q-R7-I-L-D-P_(R)-H-V-R-R-V-W-R-NH2 x B-2x 5 mol % Hoveyda-GrubbsII, at 60° C., 2 hrs Alkyl Carbamate Staples FP-0769 FP-0770 FP-0771FP-0772 FP-0773 FP-0774 FP-0776 FP-0777Ac-P-Q-S5-I-L-D-SgN-H-V-R-R-V-W-R-NH2Ac-P-Q-S5-I-L-D-SdN-H-V-R-R-V-W-R-NH2Ac-P-Q-S5-I-L-D-SeN-H-V-R-R-V-W-R-NH2Ac-P-Q-R5-I-L-D-SgN-H-V-R-R-V-W-R-NH2 Ac-P-Q-R5-I-L-D-SdN-H-V-R-R-V-W-RNH2 Ac-P-Q-R5-I-L-D-SeN-H-V-R-R-V-W-R-NH2Ac-P-Q-R5-I-L-D-RdN-H-V-R-R-V-W-R-NH2Ac-P-Q-R5-I-L-D-ReN-H-V-R-R-V-W-R-NH2             x x x x x x x x x x x

FP-0778 FP-0779 FP-0780 FP-0782 FP-0783 FP-0785 FP-0786Ac-P-Q-SgN-I-L-D-S5-H-V-R-R-V-W-R-NH2Ac-P-Q-SdN-I-L-D-S5-H-V-R-R-V-W-R-NH2Ac-P-Q-SeN-I-L-D-S5-H-V-R-R-V-W-R-NH2Ac-P-Q-RdN-I-L-D-R5-H-V-R-R-V-W-R-NH2Ac-P-Q-ReN-I-L-D-R5-H-V-R-R-V-W-R-NH2Ac-P-Q-RdN-I-L-D-S5-H-V-R-R-V-W-R-NH2Ac-P-Q-ReN-I-L-D-S5-H-V-R-R-V-W-R-NH2 x x x x x           x x

TABLE 5  Exemplary FP EC₅₀ data (nM) (Table 5 disclolses SEQ ID NOS 229, 231, 232, 234-238, 240, 242,244, 334, 246, 248, 250, 252, 253, 339, 256, 257, 259, 260, 261,263, 265, 266, 268, 270-274, 228, 230, 233, 239, 241, 243, 245, 247, 249, 251, 254, 255, 258, 262, 264, 267, 269 and 275). Peptide IDSequence FP EC₅₀ (nM)  Azetidine Carbamate Staples FP-0721cAc-P-Q-Az-I-L-D-R4-H-V-R-R-V-W-R-NH2 1300 FP-0723cAc-P-Q-Az-I-L-D-R5-H-V-R-R-V-W-R-NH2 1400 FP-0724cAc-P-Q-Az-I-L-D-S5-H-V-R-R-V-W-R-NH2 540 FP-0725cAc-P-Q-Az-I-L-D-R6-H-V-R-R-V-W-R-NH2 80 FP-0727cAc-P-Q-Az-I-L-D-R7-H-V-R-R-V-W-R-NH2 520 FP-0728cAc-P-Q-Az-I-L-D-S7-H-V-R-R-V-W-R-NH2 2100 FP-0731cAc-P-Q-R4-I-L-D-Az-H-V-R-R-V-W-R-NH2 4700 FP-0733cAc-P-Q-R5-I-L-D-Az-H-V-R-R-V-W-R-NH2 920 FP-0734cAc-P-Q-S5-I-L-D-Az-H-V-R-R-V-W-R-NH2 >5000 FP-0735cAc-P-Q-R6-I-L-D-Az-H-V-R-R-V-W-R-NH2 1600 FP-0736cAc-P-Q-S6-I-L-D-Az-H-V-R-R-V-W-R-NH2 890 FP-0737cAc-P-Q-R7-I-L-D-Az-H-V-R-R-V-W-R-NH2 TBD FP-0738cAc-P-Q-S7-I-L-D-Az-H-V-R-R-V-W-R-NH2 1800 Pyrrolidine Carbamate StaplesFP-0743c Ac-P-Q-P _(R)-I-L-D-R5-H-V-R-R-V-W-R-NH2 740 FP-0745c Ac-P-Q-P_(R)-I-L-D-R6-H-V-R-R-V-W-R-NN2  110 FP-0751c Ac-P-Q-P_(S)-I-L-D-S5-H-V-R-R-V-W-R-NH2 2000 FP-0752c Ac-P-Q-P_(S)-I-L-D-S6-H-V-R-R-V-W-R-NH2 2600 FP-0753c Ac-P-Q-P_(S)-I-L-D-S7-H-V-R-R-V-W-R-NH2 low yield  FP-0758c Ac-P-Q-R5-I-L-D-P_(S)-H-V-R-R-V-W-R-NH2  2700 FP-0761c Ac-P-Q-S6-I-L-D-P_(S)-H-V-R-R-V-W-R-NH2  460 FP-0763c Ac-P-Q-S7-I-L-D-P_(S)-H-V-R-R-V-W-R-NH2 115 FP-0765c Ac-P-Q-R4-I-L-D-P_(R)-H-V-R-R-V-W-R-NH2 30 FP-0766c Ac-P-Q-R5-I-L-D-P_(R)-H-V-R-R-V-W-R-NH2 20 FP-0767c Ac-P-Q-R6-I-L-D-P_(R)-H-V-R-R-V-W-R-NH2 110 FP-0768c Ac-P-Q-R7-I-L-D-P_(R)-H-V-R-R-V-W-R-NH2 150 Alkyl Carbamate Staples FP-0776cAc-P-Q-R5-I-L-D-RdN-H-V-R-R-V-W-R-NH2 570 FP-0777cAc-P-Q-R5-I-L-D-ReN-H-V-R-R-V-W-R-NH2 560 FP-0778cAc-P-Q-SgN-I-L-D-S5-H-V-R-R-V-W-R-NH2 1000 FP-0779cAc-P-Q-SdN-I-L-D-S5-H-V-R-R-V-W-R-NH2 1300 FP-0780cAc-P-Q-SeN-I-L-D-S5-H-V-R-R-V-W-R-NH2 1700 FP-0782cAc-P-Q-RdN-I-L-D-R5-H-V-R-R-V-W-R-NH2 380 FP-0783cAc-P-Q-ReN-I-L-D-R5-H-V-R-R-V-W-R-NH2 990 Amino Staples (Cyclic) FP-0721Ac-P-Q-Az-I-L-D-R4-H-V-R-R-V-W-R-NH2  TBD FP-0723Ac-P-Q-Az-I-L-D-R5-H-V-R-R-V-W-R-NH2 TBD FP-0725Ac-P-Q-Az-I-L-D-R6-H-V-R-R-V-W-R-NH2  580 FP-0734Ac-P-Q-S5-I-L-D-Az-H-V-R-R-V-W-R-NH2  >5000 FP-0735Ac-P-Q-R6-I-L-D-Az-H-V-R-R-V-W-R-NH2  2360 FP-0736Ac-P-Q-S6-I-L-D-Az-H-V-R-R-V-W-R-NH2  470 FP-0738Ac-P-Q-S7-I-L-D-Az-H-V-R-R-V-W-R-NH2  200 FP-0743 Ac-P-Q-P_(R)-I-L-D-R5-H-V-R-R-V-W-R-NH2 >5000 FP-0745 Ac-P-Q-P_(R)-I-L-D-R6-H-V-R-R-V-W-R-NH2 210 FP-0751 Ac-P-Q-P_(S)-I-L-D-S5-H-V-R-R-V-W-R-NH2 >5000 FP-0753 Ac-P-Q-P_(S)-I-L-D-S7-H-V-R-R-V-W-R-NH2 >5000 FP-0758 Ac-P-Q-R5-I-L-D-P_(S)-H-V-R-R-V-W-R-NH2 >5000 FP-0763 Ac-P-Q-S7-I-L-D-P_(S)-H-V-R-R-V-W-R-NH2 1110 FP-0767 Ac-P-Q-R6-I-L-D-P_(R)-H-V-R-R-V-W-R-NH2 700 FP-0768 Ac-P-Q-R7-I-L-D-P_(R)-H-V-R-R-V-W-R-NH2 >5000 Amino Staples (Acyclic)  FP-0776Ac-P-Q-R5-I-L-D-RdN-H-V-R-R-V-W-R-NH2 580 FP-0777Ac-P-Q-R5-I-L-D-ReN-H-V-R-R-V-W-R-NH2 4750 FP-0783Ac-P-Q-ReN-I-L-D-R5-H-V-R-R-V-W-R-NH2 1400 Control: FP-0787 (EC₅₀)~100nM.

TABLE 6  Exemplary amino staple formation results(Table 6 discloses SEQ ID NOS 228, 230, 334, 233, 345, 346, 239, 241, 243,347, 245, 249, 251, 348, 255, 349, 298, 258, 350, 351, 262, 264, 267, 269,309-311, 313 and 275). Multiple Peptide Reaction by Double ID SequenceComplete Incomplete products Isomer Amino Staple (Cyclic) FP-0721Ac-P-Q-Az-I-L-D-R4-H-V-R-R-V-W-R-NH2 no info  no info no info FP-0723Ac-P-Q-Az-I-L-D-R5-H-V-R-R-V-W-R-NH2 x FP-0724Ac-P-Q-Az-I-L-D-S5-H-V-R-R-V-W-R-NH2  x x FP-0725Ar-P-Q-Az-I-L-D-R6-H-V-R-R-V-W-R-NH2 x FP-0731Ac-P-Q-R4-I-L-D-Az-H-V-R-R-V-W-R-NH2 x x x FP-0733Ac-P-Q-R5-I-L-D-Az-H-V-R-R-V-W-R-NH2 x x FP-0734Ac-P-Q-S5-I-L-D-Az-H-V-R-R-V-W-R-NH2 x FP-0735Ac-P-Q-R6-I-L-D-Az-H-V-R-R-V-W-R-NH2 x FP-0736Ac-P-Q-S6-I-L-D-Az-H-V-R-R-V-W-R-NH2 x FP-0737Ac-P-Q-R7-I-L-D-Az-H-V-R-R-V-W-R-NH2 x FP-0738Ac-P-Q-S7-I-L-D-Az-H-V-R-R-V-W-R-NH2 x FP-0743 Ac-P-Q-P_(R)-I-L-D-R5-H-V-R-R-V-W-R-NH2 x FP-0745 Ac-P-Q-P_(R)-I-L-D-R6-H-V-R-R-V-W-R-NH2 x FP-0751 Ac-P-Q-P_(S)-I-L-D-S5-H-V-R-R-V-W-R-NH2 x FP-0752 Ac-P-Q-P_(S)-I-L-D-S6-H-V-R-R-V-W-R-NH2 x x FP-0758 Ac-P-Q-R5-I-L-D-P_(S)-H-V-R-R-V-W-R-NH2 x fp-0759 Ac-P-Q-S5-I-L-D-P_(S)-H-V-R-R-V-W-R-NH2 x x FP-0761 Ac-P-Q-S6-I-L-D-P_(S)-H-V-R-R-V-W-R-NH2 x FP-0763 Ac-P-Q-S7-I-L-D-P_(S)-H-V-R-R-V-W-R-NH2 x FP-0765 Ar-P-Q-R4-I-L-D-P_(R)-H-V-R-R-V-W-R-NH2 x x FP-0766 Ac-P-Q-R5-I-L-D-P_(R)-H-V-R-R-V-W-R-NH2 x x FP-0767 Ac-P-Q-R6-I-L-D-P_(R)-H-V-R-R-V-W-R-NH2 x FP-0768 Ac-P-Q-R7-I-L-D-P_(R)-H-V-R-R-V-W-R-NH2 x Amino Staples (Acyclic) FP-0776Ac-P-Q-R5-I-L-D-RdN-H-V-R-R-V-W-R-NH2 x FP-0771Ac-P-Q-R5-I-L-D-ReN-H-V-R-R-V-W-R-NH2 x Fp-0778Ac-P-Q-SgN-I-L-D-S5-H-V-R-R-V-W-R-NH2 x x FP-0779Ac-P-Q-sdN-I-L-D-S5-H-V-R-R-V-W-R-NH2 x FP-0780Ac-P-Q-SeN-I-L-D-S5-H-V-R-R-V-W-R-NH2 x FP-0782Ac-P-Q-RdN-I-L-D-R5-H-V-R-R-V-W-R-NH2 x FP-0183Ac-P-Q-ReN-I-L-D-R5-I-V-R-R-V-W-R-NH2 x

While various embodiments have been described and illustrated herein,those of ordinary skill in the art will readily envision a variety ofother means and/or structures for performing the functions and/orobtaining the results and/or one or more of the advantages described inthe present disclosure, and each of such variations and/or modificationsis deemed to be included. More generally, those skilled in the art willreadily appreciate that all parameters, dimensions, materials, andconfigurations described herein are meant to be example and that theactual parameters, dimensions, materials, and/or configurations willdepend upon the specific application or applications for which theteachings of the present disclosure is/are used. Those skilled in theart will recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of thedisclosure described in the present disclosure. It is, therefore, to beunderstood that the foregoing embodiments are presented by way ofexample only and that, provided technologies, including those to beclaimed, may be practiced otherwise than as specifically described andclaimed. In addition, any combination of two or more features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the scope of the present disclosure.

The invention claimed is:
 1. A peptide comprising:[X¹]_(p1)[X²]_(p2)—X³X⁴X⁵X⁶X⁷X⁸X⁹X¹⁰—[X¹¹]_(p11)[X¹²]_(p12)[X¹³]_(p13),wherein: each of p1, p2, p11, p12 and p13 is independently 0 or 1; eachof X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², and X¹³ isindependently an amino acid residue; at least two of X¹, X², X³, X⁴, X⁵,X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², and X¹³ comprise side chains that arelinked together to form a staple, wherein the peptide comprises astructure selected from:

wherein each X is independently an amino acid residue.
 2. The peptide ofclaim 1, wherein the peptide comprises


3. The peptide of claim 1, wherein the peptide comprises


4. The peptide of claim 1, wherein the peptide comprises


5. The peptide of claim 1, wherein the peptide comprises


6. The peptide of claim 1, wherein the peptide comprises


7. The peptide of claim 1, wherein the peptide comprises


8. The peptide of claim 1, wherein the peptide comprises


9. The peptide of claim 1, wherein the peptide comprises


10. The peptide of claim 1, wherein the peptide comprises


11. The peptide of claim 1, wherein the peptide comprises one and nomore than one staple.
 12. The peptide of claim 1, wherein the peptidecomprises a structure selected from:

and wherein the peptide comprises two or more staples.
 13. The peptideof claim 12, wherein at least two staples are bonded to the same peptidebackbone atom.
 14. The peptide of claim 12, wherein no two staples arebonded to the same peptide backbone atom.
 15. A peptide comprising:[X¹]_(p1)[X²]_(p2)—X³X⁴X⁵X⁶X⁷X⁸X⁹X¹⁰—[X¹¹]_(p11)[X¹²]_(p12)[X¹³]_(p13),wherein: each of p1, p2, p11, p12 and p13 is independently 0 or 1; eachof X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², and X¹³ isindependently an amino acid residue; at least two of X¹, X², X³, X⁴, X⁵,X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², and X¹³ comprise side chains that arelinked together to form a staple; and the peptide comprises two or morestaples, wherein the peptide comprises a structure selected from:

wherein each X is independently an amino acid residue.
 16. The peptideof claim 15, wherein at least two staples are bonded to the same peptidebackbone atom.
 17. The peptide of claim 15, wherein no two staples arebonded to the same peptide backbone atom.
 18. A peptide having thestructure of:

or a salt thereof, wherein each of R^(a), R¹, R², R³, and R⁴ isindependently R′; R^(b) is R′, —OR′ or —N(R′)₂; each X is independentlyan amino acid residue; a is 0-20; each of b, c, s, and d isindependently 1-20; each of C¹ and C² is independently a carbon atom;each L^(s) is independently -L^(s1)-L^(s2)-L^(s3)-, wherein L^(s1) isbonded to C¹ and L^(s3) is bonded to C²; each of L^(s1), L^(s2), andL^(s3) is independently L; each L is independently a covalent bond, oran optionally substituted, bivalent C₁-C₂₀ aliphatic group wherein oneor more methylene units of the aliphatic group are optionally andindependently replaced with —C(R′)₂—, -Cy-, —O—, —S—, —S—S—, —N(R′)—,—C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)O—,—S(O)—, —S(O)₂—, —S(O)₂N(R′)—, —C(O)S—, or —C(O)O—; each -Cy- isindependently an optionally substituted bivalent group selected from aC₃₋₂₀ cycloaliphatic ring, a C₆₋₂₀ aryl ring, a 5-20 membered heteroarylring having 1-10 heteroatoms independently selected from oxygen,nitrogen, sulfur, phosphorus and silicon, and a 3-20 memberedheterocyclyl ring having 1-10 heteroatoms independently selected fromoxygen, nitrogen, sulfur, phosphorus and silicon; each R′ isindependently —R, —C(O)R, —CO₂R, or —SO₂R; each R is independently —H,or an optionally substituted group selected from C₁₋₃₀ aliphatic, C₁₋₃₀heteroaliphatic having 1-10 heteroatoms independently selected fromoxygen, nitrogen, sulfur, phosphorus and silicon, C₆₋₃₀ aryl, C₆₋₃₀arylaliphatic, C₆₋₃₀ arylheteroaliphatic having 1-10 heteroatomsindependently selected from oxygen, nitrogen, sulfur, phosphorus andsilicon, 5-30 membered heteroaryl having 1-10 heteroatoms independentlyselected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30membered heterocyclyl having 1-10 heteroatoms independently selectedfrom oxygen, nitrogen, sulfur, phosphorus and silicon, or two R groupsare optionally and independently taken together to form a covalent bond,or: two or more R groups on the same atom are optionally andindependently taken together with the atom to form an optionallysubstituted, 3-30 membered, monocyclic, bicyclic or polycyclic ringhaving, in addition to the atom, 0-10 heteroatoms independently selectedfrom oxygen, nitrogen, sulfur, phosphorus and silicon; or two or more Rgroups on two or more atoms are optionally and independently takentogether with their intervening atoms to form an optionally substituted,3-30 membered, monocyclic, bicyclic or polycyclic ring having, inaddition to the intervening atoms, 0-10 heteroatoms independentlyselected from oxygen, nitrogen, sulfur, phosphorus and silicon; whereinthe peptide comprises two or more staples, wherein the peptide comprisesa structure selected from:


19. A pharmaceutical composition comprising a peptide of claim 1 or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 20. A pharmaceutical composition comprising apeptide of claim 15 or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.
 21. A pharmaceutical compositioncomprising a peptide of claim 18 or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier.
 22. The peptide ofclaim 1, wherein the peptide comprises


23. The peptide of claim 1, wherein the peptide comprises